EP2405019A1 - Verfahren, Reagenzien, Kits und Vorrichtung zur Proteinfunktionsanalyse - Google Patents

Verfahren, Reagenzien, Kits und Vorrichtung zur Proteinfunktionsanalyse Download PDF

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EP2405019A1
EP2405019A1 EP11154630A EP11154630A EP2405019A1 EP 2405019 A1 EP2405019 A1 EP 2405019A1 EP 11154630 A EP11154630 A EP 11154630A EP 11154630 A EP11154630 A EP 11154630A EP 2405019 A1 EP2405019 A1 EP 2405019A1
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Prior art keywords
amino acid
binding
solid phase
protein
proteins
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EP11154630A
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English (en)
French (fr)
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John H. Kenten
Hans Biebuyck
Ilia Davydov
Nisar Pampori
Steven Yan Cheng
Stefani Nelson
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Meso Scale Technologies LLC
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Meso Scale Technologies LLC
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5302—Apparatus specially adapted for immunological test procedures
    • G01N33/5304—Reaction vessels, e.g. agglutination plates
    • C—CHEMISTRY; METALLURGY
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
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Definitions

  • This invention provides apparatus, systems, system components, methods, compositions, and reagents for determining the function and activity of peptides and proteins and for identifying and characterizing molecules that affect these functions and activities.
  • the methods and reagents of the invention are used to determine the various activities of peptides and proteins including their binding specificity, binding activity, their enzymatic activity and their ability to act as substrates for enzymes.
  • This invention is especially suited for the analysis of peptide and protein function where large numbers of peptides and proteins need to be analyzed.
  • the steps involved in the transcription and translation (expression) of genes in cells are complex but the basic steps for protein to be produced from DNA are transcription and translation.
  • the DNA is first transcribed into RNA, and then the RNA is translated by the interaction of various cellular components into protein.
  • transcription and translation are coupled, meaning that RNA is translated into protein during the time that it is being transcribed from the DNA.
  • eukaryotic cells the two activities are separate, making the overall process more complicated.
  • DNA is transcribed into RNA inside the nucleus of the cell, but the RNA is further processed into mRNA and then transported outside the nucleus to the cytoplasm where it is translated into protein.
  • In vitro transcription systems using prokaryotic or eukaryotic cells are available; however, these systems are difficult to work with since intact cells are used.
  • In vitro cell-free systems are made from cell-free extracts produced from prokaryotic or eukaryotic cells that contain all the necessary components to translate DNA or RNA into protein.
  • Cell-free extracts can be prepared from prokaryotic cells such as E. coli and from eukaryotic cells such as rabbit reticulocytes and wheat germ. Cell-free systems are very popular because there are standard protocols available for their preparation and because they are commercially available from a number of sources.
  • in vitro systems have many advantages to major draw back is that the amounts of protein produced is generally low which makes the analysis of protein function and activity difficult. The problems with low levels of protein production have forced researchers to develop complex assay systems using gel based systems and radioactivity that limit the application of these in vitro methods for studies of protein function and activity.
  • E. coli S30 cell-free extracts were first described by Zubay, G. (1973 Ann. Rev. Genet. Vol 7, p. 267 ). These extracts can be used when the gene to be expressed has been cloned into a vector containing the appropriate prokaryotic regulatory sequences, such as a promoter and ribosome-binding site. Prokaryotic E. coli cell-free systems are considered coupled because transcription and translation occur simultaneously after the addition of DNA to the extract.
  • Rabbit reticulocyte lysate was described by Pelham, H. R. B. and Jackson, R. J. (1976, Eur. J. Biochem. Vol. 67, p. 247 ). This expression system is probably the most widely used cell-free system for in vitro translation, and is used in the identification of mRNA species, the characterization of their products and the investigation of transcriptional and translational control.
  • Wheat germ extract was described by Roberts, B. E. and Paterson, B. M. (1973, Proc. Natl. Acad. Sci. U.S.A., Vol. 70, P. 2330 ).
  • Cell-free extracts of wheat germ support the translation in vitro of a wide variety of viral and other prokaryotic RNAs, as well as eukaryotic mRNAs. ( Anderson, C., et al. (1983) Meth. Enzymol. 101, 635 ).
  • Post-translational modifications that have been observed in rabbit reticulocyte lysate or wheat germ extract include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis myristoylation, protein folding and proteolytic processing ( Glass, C. A. and Pollard, K. M. (1990). Promega Notes 26 ). Some modifications or processing events have required the introduction of additional biological machinery. For example, processing events, such as signal peptide cleavage and core glycosylation, are examined by adding canine microsomal membranes ( Walter, P. and Blobel, G. (1983) Meth. Enzymol. 96, 84 ) ( Walter, P. and Blobel, G. (1983) Meth. Enzymol.
  • RNA for translational studies can be obtained by either isolating mRNA or by making in vitro RNA transcripts from DNA that has been cloned into a vector containing an RNA polymerase promoter.
  • the first method isolates mRNA directly from cells.
  • the second obtains RNA for in vitro translation by in vitro transcription.
  • In vitro transcription of cloned DNA behind phage polymerase promoters was described by Krieg, P. and Melton, D (1984, Nucl. Acids Res., Vol. 12, p. 7057 ). This method has become a standard method for obtaining RNA from cloned genes for use in in vitro translation reactions.
  • the method uses DNA or a gene of interest that is cloned into a vector containing a promoter for an RNA polymerase.
  • the vector is then purified and followed by an in vitro transcription reaction to make RNA transcripts,
  • a number of vectors containing the SP6, T7 and T3 RNA polymerase promoters are commercially available and are widely used for cloning DNA.
  • Continuous translation involves a bioreactor (such as an Amicon 8MC ultrafiltration unit) in which large scale reactions are set up and protein is continually translated over extended periods of time.
  • the reaction requires that a buffer be fed into the reaction as it progresses, and also requires that the products of translation be removed from the reaction filter unit.
  • This type of system works well with E. coli S30 extract and wheat germ extract when RNA template is introduced. See Spirin, et al. (1988) Science, Vol 242, 1162-1164 .
  • the system also works using RNA templates in rabbit reticulocyte lysate. See Ryabova, et al. (1989) Nucl, Acid Res., Vol.
  • the proteins from these translation or transcription translation reactions have been labeled using a variety of methods. This step is important given the fact that very small amounts of protein are produced in these translation reactions relative to the total amount of protein (4-0.4 ug/ml relative to 50-60 mg/ml of endogenous proteins).
  • One labeling method is the genetic modification of the gene or genes of interest, which results in the translation of a protein containing amino acids, amino acid sequences or post-translational modifications not found in the normal translation product of the gene. These modifications provide for the use of various methods for detection and purification of the translated proteins. These methods for the modification of the normal gene product are not always helpful as they can prevent the normal function of the protein due to the modification of the N terminal or C terminal portions of the protein. Also these modifications can prove to be costly and time consuming when applied on large scale.
  • Radioactive labeled amino acid sequences produced this way have been valuable in the study, detection and determination of these protein function and properties.
  • the problems that are associated with the use of radioactivity are well known and make its use on a large scale problematic, unsafe and costly.
  • ECL labels include: i) organometallic compounds where the metal is from, for example, the noble metals of group VIII, including Ru-containing and Os-containing organometallic compounds such as the tris-bipyridyl-ruthenium (RuBpy) moiety and ii) luminot and related compounds.
  • ECL coreactants Species that participate with the ECL label in the ECL process are referred to herein as ECL coreactants.
  • Commonly used coreactants include tertiary amines (e,g., see U.S. Patent No. 5,846,485 ), oxalate, and persulfate for ECL from RuBpy and hydrogen peroxide for ECL from luminol (see, e.g., U.S. Patent No. 5,240,863 .
  • the light generated by ECL labels can be used as a reporter signal in diagnostic procedures ( Bard et al., U.S. Patent No. 5,238,808 ).
  • an ECL label can be covalently coupled to a binding agent such as an antibody, nucleic acid probe, receptor or ligand; the participation of the binding reagent in a binding interaction can be monitored by measuring ECL emitted from the ECL label.
  • the ECL signal from an ECL-active compound may be indicative of the chemical environment (see, e.g., U.S. Patent No. 5,641,623 which describes ECL assays that monitor the formation or destruction of ECL coreactants).
  • ECL labels, ECL assays and instrumentation for conducting ECL assays see U.S. Patents Nos.
  • ECL instruments have demonstrated exceptional performance. They have become widely used for reasons including their excellent sensitivity, dynamic range, precision, and tolerance of complex sample matrices.
  • the commercially available instrumentation uses flow cell-based designs with permanent reusable flow cells.
  • ECL instrumentation has been disclosed that uses reagents immobilized on the electrode used to induce ECL (see, e,g., U.S. Patent No. 6,207,369 and Published PCT Application No. WO98/12539 ).
  • Multi-well plates having integrated electrodes suitable for such ECL measurements have also been recently disclosed (see, e.g., copending Provisional Application No.
  • multi-well assay plates allow for the parallel processing and analysis of multiple samples distributed in multiple wells of a plate.
  • samples and reagents are stored, processed and/or analyzed in multi-well assay plates (also known as microplates or microtiter plates).
  • Multi-well assay plates can take a variety of forms, sizes and shapes. For convenience, some standards have appeared for some instrumentation used to process samples for high throughput assays. Assays carried out in standardized plate formats can take advantage of readily available equipment for storing and moving these plates as well as readily available equipment for rapidly dispensing liquids in and out of the plates.
  • Some well established multi-well plate formats include those found on 96-well plates (12 x 8 array of wells), 384-well plates (24 x 16 array of wells) and 1536-well plate (48 x 32 array of well).
  • the Society for Biomolecular Screening has published recommended microplate specifications for a variety of plate formats (see, http://www.sbsonline.org), the recommended specifications hereby incorporated by reference.
  • This invention provides apparatus, systems, system components, methods, compositions, and reagents for determining the function and activity of peptides and proteins and for identifying and characterizing molecules that affect these functions and activities.
  • the methods and reagents of the invention are used to determine the various activities of peptides and proteins including their binding specificity, binding activity, their enzymatic activity and their ability to act as substrates for enzymes.
  • This invention is especially suited for the analysis of peptide and protein function where large numbers of peptides and proteins need to be analyzed.
  • One embodiment of the invention comprises a multiwell plate with a plurality of wells, preferably with at least 20 wells containing the in vitro transcription and translation products of at least one, and preferably plurality of, unique nucleic acid construct.
  • the multiwell plate may advantageously have at least 40 wells or most advantageously at least 84 wells.
  • the multi-well assay plates may have any number of wells of any size or shape, arranged in any pattern or configuration, and can be composed of a variety of different materials.
  • Preferred embodiments of the invention use industry standard formats for the number, size, shape and configuration of the plate and wells. Examples of standard formats include 96-, 384-, 1536-, and 9600-well plates, with the wells configured in two-dimensional arrays.
  • Other formats may include single well plates (preferably having a plurality of assay domains), 2 well plates, 6 well plates, 24 well plates, and 6144 well plates.
  • the number of unique nucleic acid constructs advantageously is at least 2, more preferably at least 6, even more preferably at least 24, even more preferably at least 40, even more preferably at least 84 and most advantageously at least 300.
  • the wells of the multiwell plate can comprise carbon or a carbon composite materials which may be used as solid phases in solid phase assays.
  • the wells of the multiwell plate may include integrated electrodes which may also be used as solid phases in solid phase assays, preferably these are configured so that the multiwell plate is able to support electrochemiluminescence.
  • each well contains multiple zones or spots which allow multiple amino acid sequences containing the modified amino acids to be individually contacted to one of the multiple zones or spots in a well of a multiwell plate.
  • amino acid sequences may be contacted with multiple zones in a well, e.g., to measure multiple different characteristics of an amino acid sequence.
  • the invention also comprises a multiwell plate with at least 20 wells containing the in vitro transcription and translation products of at least one unique nucleic acid construct and coated with the products of the in vitro transcription translation reaction.
  • the multiwell plate may advantageously have at least 40 wells or most advantageously at least 84 wells.
  • the multi-well assay plates may have any number of wells of any size or shape, arranged in any pattern or configuration, and can be composed of a variety of different materials.
  • Preferred embodiments of the invention use industry standard formats for the number, size, shape and configuration of the plate and wells. Examples of standard formats include 96-, 384-, 1536-, and 9600-well plates, with the wells configured in two-dimensional arrays.
  • Other formats may include single well plates (preferably having a plurality of assay domains), 2 well plates, 6 well plates, 24 well plates, and 6144 well plates.
  • the number of unique nucleic acid constructs advantageously is at least 2, more preferably at least 6, even more preferably at least 24, even more preferably at least 40, even more preferably at least 84 and most advantageously at least 300.
  • the wells of the multiwell plate can comprise carbon or a carbon composite materials which may be used as solid phases in solid phase assays.
  • the wells of the multiwell plate may include integrated electrodes which may also be used as solid phases in solid phase assays, preferably these are configured so that the multiwell plate is able to support electrochemiluminescence.
  • each well contains multiple zones or spots which allow multiple amino acid sequences containing the modified amino acids each individually to be contacted to one of the multiple zones or spots in a well of a multiwell plate.
  • amino acid sequences may be contacted with multiple zones in a well, e.g., to measure multiple different characteristics of an amino acid sequence.
  • the invention comprises a multiwell plate containing beads with each well containing the in vitro transcription and translation products of at least one unique nucleic acid construct.
  • the multiwell plate may advantageously have magnetic beads.
  • the in vitro transcription and translation is carried out in the presence of a tRNA precharged with a modified amino acid.
  • the invention also comprises a multi-well plate with at least 20 wells coated with a specific binding species that is used to bind the in vitro transcription and translation products of at least one unique nucleic acid construct.
  • the invention also comprises these plates, wherein the well surfaces provide a solid phase which coated with the products of the in vitro transcription translation reaction, preferably via binding to the specific binding species.
  • the multiwell plate may advantageously have at least 40 wells or most advantageously at least 84 wells. Also the number of unique nucleic acid constructs advantageously is at least 40 and most advantageously at least 84.
  • the wells of the multiwell plate can comprise carbon or a carbon composite materials which may be used as solid phases in solid phase assays and/or to support the immobilized amino acid sequences.
  • the wells of the multiwell plate may include integrated electrodes which may be used as solid phases in solid phase assays and/or to support the immobilized amino acid sequences, preferably these are configured so that the multiwell plate is able to support electrochemiluminescence.
  • the in vitro translation is carried out in the presence of a tRNA precharged with a modified amino acid.
  • each well contains multiple zones (sometimes referred to herein as assay domains or spots) which allow multiple amino acid sequences containing the modified amino acids each individually to be contacted to one of the multiple zones or spots in a well of a multiwell plate.
  • amino acid sequences may be contacted with multiple zones in a well, e.g., to measure multiple different characteristics of an amino acid sequence.
  • the specific binding species is selected from the nucleic acid, peptides or proteins
  • the invention also comprises a multiwell plate with at least 20 wells coated with an amino acid sequence containing a modified amino acid that is produced as follows. Initially a nucleic acid construct is obtained and transcribed to generate DNA. This RNA is then translated in a cell-free translation system (preferably a cell lysate) containing a tRNA precharged with a modified amino acid, to produce an amino acid sequence containing the modified amino acid. These transcription and translation steps can also be combined in a single in vitro reaction. This amino acid sequence is then contacted with a well of a multiwell plate with at least 20 wells, where by a well surface coated with an amino acid sequence containing a modified amino acid is produced.
  • a cell-free translation system preferably a cell lysate
  • the multiwell plate can contain at least 40 wells most advantageously the multiwell plate has at least 84 wells.
  • the wells of the multiwell plate can comprise carbon or a carbon composite materials which may be used as solid phases in solid phase assays and/or to support the immobilized amino acid sequences.
  • the wells of the multiwell plate may' include integrated electrodes which may be used as solid phases in solid phase assays and/or to support the immobilized amino acid sequences, preferably these are configured so that the multiwell plate is able to support electrochemiluminescence.
  • each well contains multiple zones or spots which allow multiple amino acid sequences containing the modified amino acids each individually to be contacted to one of the multiple zones or spots in a well of a multiwell plate.
  • amino acid sequences may be contacted with multiple zones in a well, e.g., to measure multiple different characteristics of an amino acid sequence.
  • the invention also comprises a method of producing a multiwell plate with at least 20 wells coated with an amino acid sequence containing a modified amino acid.
  • This is, preferably, produced by obtaining a nucleic acid construct and transcribing it to generate RNA.
  • This RNA is then translated in a cell-free translation system containing a tRNA precharged with a modified amino acid, to produce an amino acid sequence containing a modified amino acid.
  • transcription and translation steps can also be combined in a single in vitro reaction.
  • Amino acid sequences prepared in this way are then introduced into wells of a multiwell plate with at least 20 wells, where by a multiwell plate having wells coated with these amino acid sequences is produced.
  • the wells of the multiwell plate can comprise carbon or a carbon composite materials which may be used as solid phases in solid phase assays and/or to support the immobilized amino acid sequences.
  • the wells of the multiwell plate may include integrated electrodes which may be used as solid phases in solid phase assays and/or to support the immobilized amino acid sequences, preferably these are configured so that the multi-well plate is able to support electrochemiluminescence.
  • each well contains multiple zones or spots which allow multiple amino acid sequences containing the modified amino acids each individually to be contacted to one of the multiple zones or spots in a well of a multiwell plate.
  • amino acid sequences may be contacted with multiple zones in a well, e.g., to measure multiple different characteristics of an amino acid sequence.
  • the invention also comprises a method of producing a multiwell plate with at least 20 wells coated with an amino acid sequence containing a modified amino acid and post-translational modifications. This is, preferably, produced by obtaining one or a series of nucleic acid constructs followed by transcribing these to generate RNA. These RNA are then translated in a cell-free system (preferably a cell lysate) containing a tRNA precharged with a modified amino acid, to produce an amino acid sequence containing said modified amino acid and post translational modifications. These amino acid sequences are then introduced into wells of a multiwell plate with at least 20 wells, where by a multiwell plate coated with an amino acid sequence containing a modified amino acid and post-translational modifications is produced.
  • the wells of the multiwell plate can comprise carbon or a carbon composite materials which may be used as solid phases in solid phase assays and/or to support the immobilized amino acid sequences.
  • the wells of the multiwell plate may include integrated electrodes which may be used as solid phase supports in solid phase assays and/or to support the immobilized amino acid sequences, preferably these are configured so that the multiwell plate is able to support electrochemiluminescence.
  • each well contains multiple zones or spots which allow multiple amino acid sequences containing the modified amino acids each individually to be contacted to one of the multiple zones or spots in a well of a multiwell plate.
  • amino acid sequences may be contacted with multiple zones in a well, e.g., to measure multiple different characteristics of an amino acid sequence.
  • the invention also comprises a method of producing a multiwell plate with at least 20 wells coated with an amino acid sequence containing a modified amino acid and post-translational modifications. This is, preferably, produced by obtaining one or a series of nucleic acid constructs followed by transcribing these to generate RNA. These RNAs are then translated in a cell-free system (preferably a cell lysate) containing a tRNA precharged with a modified amino acid, to produce an amino acid sequence containing said modified amino acid. These amino acid sequences are then introduced into wells of a multiwell plate with at least 40 wells, to coat said wells with amino acid sequences containing a modified amino acid.
  • a multiwell plate with at least 40 wells coated with an amino acid sequence containing a modified amino acid and a post-translational modification is produced.
  • the wells of the multiwell plate can comprise carbon or a carbon composite materials which may be used as solid phases in solid phase assays.
  • the wells of the multiwell plate may include integrated electrodes, preferably these are configured so that the multiwell plate is able to support electrochemiluminescence.
  • each well contains multiple zones or spots which allow multiple amino acid sequences containing the modified amino acids each individually to be contacted to one of the multiple zones or spots in a well of a multiwell plate.
  • amino acid sequences may be contacted with multiple zones in a well, e.g., to measure multiple different characteristics of an amino acid sequence.
  • the invention also comprises a method of producing a multiwell plate with at least 20 wells coated with an amino acid sequence containing a modified amino acid and post-translational modifications.
  • This is, preferably, produced by obtaining one or a series of nucleic acid constructs and transcribing these nucleic acids to generate RNA.
  • the RNA is then translated in a cell-free translation system (preferably a cell lysate) containing a tRNA precharged with a modified amino acid, to produce an amino acid sequence containing said modified amino acid.
  • a cell-free translation system preferably a cell lysate
  • These amino acid sequences are then introduced into the wells of a multiwell plate with at least 20 wells, to coat said multiwell plate with an amino acid sequence containing a modified amino acid.
  • coated multiwell plates are then contacted with an enzyme effective at removing a post-translational modification.
  • an enzyme effective at removing a post-translational modification the multiwell plate is contacted with a second enzyme, where by a multiwell plate with at least 20 wells coated with an amino acid sequence containing a modified amino acid and a post-translational modification is produced.
  • the wells of the multiwell plate can comprise carbon or a carbon composite materials which may be used as solid phases in solid phase assays and/or to support the immobilized amino acid sequences.
  • the wells of the multiwell plate may include integrated electrodes which may be used as solid phase supports in solid phase assays and/or to support the immobilized amino acid sequences, preferably these are configured so that the multiwell plate is able to support electrochemiluminescence.
  • each well contains multiple zones or spots which allow multiple amino acid sequences containing the modified amino acids each individually to be contacted to one of the multiple zones or spots in a well of a multiwell plate.
  • amino acid sequences may be contacted with multiple zones in a well, e.g., to measure multiple different characteristics of an amino acid sequence.
  • the invention also comprises a method for screening nucleic acid constructs for those encoding amino acid sequences subject to post-translational modifications. This is, preferably, achieved as follows.
  • a nucleic acid construct is obtained and transcribed to generate RNA.
  • This RNA is then translated in a cell-free translation system containing a tRNA precharged with a modified amino acid, to produce an amino acid sequence containing said modified amino acid.
  • This amino acid sequence is then subjected to the detection of its post-translational modifications using, preferably, a solid phase assay.
  • the amino acid sequence is captured onto a solid phase using a binding species specific for the post-translational modification. Following this the captured amino acid sequences is detected on the solid phase using the modified amino acid.
  • amino acid sequence is captured onto a solid phase via a binding species specific for said modified amino acid.
  • This captured amino acid sequence is then subject to detection using, e.g., a binding species specific for the post-translational modification.
  • Amino acid sequences having the desired activity may be identified from their nucleic acid sequence.
  • the invention also comprises a method for screening nucleic acid constructs for those encoding amino acid sequences subject to post-translational modifications. This is, preferably, achieved as follows. A nucleic acid construct is obtained and transcribed to generate RNA. This RNA is then translated in a cell-free translation system containing a tRNA precharged with a modified amino acid and a substrate for a post-translational modification, to produce an amino acid sequence containing said modified amino acid and said post-translational modification. The detection of the post-translational modifications of the amino acid sequence is achieved using, preferably, a solid phase assay.
  • the amino acid sequence is captured onto a solid phase via a binding species specific for said modified amino acid and detecting using a binding species specific for a post-translational modification.
  • the amino acid sequence is captured onto a solid phase using a binding species specific for the post-translational modification. This is followed by detecting the captured amino acid sequences on the solid phase using, e.g., a binding species specific for the modified amino acid.
  • Amino acid sequences having the desired activity may be identified from their nucleic acid sequence.
  • the invention also comprises a method for screening nucleic acid constructs for those encoding amino acid sequences subject to post-translational modifications. This is, preferably, achieved as follows.
  • a nucleic acid construct is obtained and transcribed to generate RNA.
  • the RNA is translated in a cell-free translation system containing a tRNA precharged with a modified amino acid and a substrate for a post-translational modification modified with a label, to produce an amino acid sequence containing the modified amino acid and the post-translational modification.
  • the post-translational modifications of the amino acid sequence are, preferably, detected using a solid phase assay.
  • the amino acid sequence is captured onto a solid phase via a binding species specific for said modified amino acid and detected using the label on the post-translational modification.
  • the amino acid sequence is captured onto a solid phase via a binding species for the label on the post-translational modification and detected using the modified amino acid.
  • Amino acid sequences having the desired activity may be identified from their nucleic acid sequence. Examples of binding species specific for post-translational modifications include antibodies, antibodies to ubiquitin and antibodies to phosphotyrosine.
  • the invention also comprises a method for screening nucleic acid constructs for those encoding amino acid sequences with enzymatic activity, preferably, comprising the following steps.
  • a nucleic acid construct is obtained and transcribed to generate RNA.
  • the RNA is translated in a cell-free system containing a tRNA precharged with a modified amino acid, to produce an amino acid sequence containing the modified amino acid.
  • the enzymatic activity of the amino acid sequence is preferably detected using a solid phase assay.
  • the amino acid sequence is captured onto a solid phase via a binding species specific for the modified amino acid, enzyme reaction buffer is added and the product of the enzyme activity is detected.
  • Amino acid sequences having the desired activity may be identified from their nucleic acid sequence.
  • the invention also comprises a method for screening nucleic acid constructs for those encoding amino acid sequences that are substrates of enzymatic activity comprising, preferably, the following steps.
  • a nucleic acid construct is obtained and transcribed to generate RNA.
  • the RNA is translated in a cell-free system containing a tRNA precharged with a modified amino acid, to produce an amino acid sequence containing the modified amino acid.
  • the amino acid sequences which are substrates of an enzyme activity are detected using, preferably, a solid phase assay.
  • the amino acid sequences are captured onto a solid phase via a binding species specific for the modified amino acid, an enzyme is added and the product of the enzyme activity is detected.
  • Amino acid sequences having the desired activity may be identified from their nucleic acid sequence.
  • the amino acid sequences are pre-treated prior to contact with the enzyme activity to add or remove a post-translational modification.
  • the invention also comprises a method for screening nucleic acid constructs for those encoding amino acid sequences with binding activity comprising, preferably, the following steps.
  • a nucleic acid construct is obtained and transcribed to generate RNA.
  • the RNA is translated in a cell-free system containing a tRNA precharged with a modified amino acid, to produce an amino acid sequence containing the modified amino acid.
  • the binding activity towards a binding partner is, preferably, detected in a solid phase assay.
  • the assay comprises capturing the amino acid sequence onto a solid phase by binding it to a binding partner immobilized on the solid phase and detecting the captured amino acid sequence using the modified amino acid.
  • the assay comprises capturing the amino acid sequence on a solid phase via a binding reagent specific for the modified amino acid and detecting the binding partner on the solid phase.
  • Amino acid sequences having the desired activity may be identified from their nucleic acid sequence.
  • the invention also comprises a method for screening nucleic acid constructs for those encoding amino acid sequences with binding activity comprising, preferably, the following steps.
  • a nucleic acid construct is obtained and transcribed to generate RNA.
  • the RNA is translated in a cell-free system containing a tRNA precharged with a modified amino acid, to produce an amino acid sequence containing the modified amino acid.
  • the binding activity of the amino acid sequence for a binding partner is measured, preferably, using a solid phase assay.
  • the assay comprises contacting the amino acid sequence with a solid phase coated with a binding species specific for the modified amino acid, contacting the amino acid sequence with the binding partner, and detecting complexes having the binding partner on the solid phases.
  • the immobilizing step may occur before, during or after, the contacting step.
  • binding partner is captured on the solid phase and the modified amino acid is detected.
  • Amino acid sequences having the desired activity may be identified from their nucleic acid sequence.
  • the invention also comprises a method for screening nucleic acid constructs for those encoding amino acid sequences with binding activity comprising, preferably, the following steps.
  • a first nucleic acid and second nucleic acid construct are obtained and transcribed to generate a first and second RNA.
  • the first RNA is translated in a cell-free system containing a tRNA precharged with a modified amino acid comprising a first binding species, to produce a first amino acid sequence containing said first binding species.
  • the second RNA is translated in a cell lysate containing a tRNA precharged with a modified amino acid containing a detectable species, to produce a second amino acid sequence containing the detectable species.
  • the binding of the two amino acid sequences is detected using, preferably, a solid phase assay.
  • the assay comprises contacting a sample of the first amino acid sequence with the second amino acid sequence and capturing the binding species on a solid phase via a second binding species and detecting the detectable species bound to the solid phase.
  • Amino acid sequences having the desired activity may be identified from their nucleic acid sequence.
  • the invention also comprises a method for screening nucleic acid constructs for those encoding Amino acid sequences with nascent binding activity comprising, preferably, the following steps.
  • a nucleic acid construct is obtained and transcribed to generate RNA.
  • the RNA is translated in a cell lysate containing a tRNA precharged with a modified amino acid, to produce an amino acid sequence containing said modified amino acid.
  • the amino acid sequence is captured onto a solid phase via a binding species specific for the modified amino acid and contacting with an enzyme to covalently modify the amino acid sequence.
  • the binding of the modified amino acid sequence to a binding partner is detected, preferably, by a solid phase assay.
  • the assay comprises contacting the amino acid sequence with a second binding species labeled with a detectable species and detecting the binding of the second binding species to the covalently modified solid phase.
  • the second binding species may be labeled with a detectable label made using a method comprising the following steps: obtaining a nucleic acid construct, and transcribing the construct to generate RNA and translating said RNA in a cell lysate containing a tRNA precharged with a second modified amino acid, to produce an amino acid sequence containing the second modified amino acid.
  • This invention provides the identification of kinase activity for proteins that had not previously been demonstrated to have in vitro kinase activity. It also comprises methods for the determination of kinase activity. These kinases that can be used for the screening of compound libraries for inhibitors or activators of these kinases. This invention also provides for assays that allow the investigation of the activity of these kinase isolated from cells and or tissue samples for both studies into disease and diagnostic applications.
  • the invention also comprises a method for screening for antigens that bind antibodies comprising, preferably, the following steps.
  • a nucleic acid construct is obtained and transcribed to generate RNA.
  • the RNA is translated in a cell-free system to produce an amino acid sequence.
  • the binding of the amino acid sequence to an antibody is, preferably, measured using a solid phase assay.
  • the amino acid sequence is immobilized on a solid phase and contacted with an antibody. Binding of the antibody to the solid phase is detected by measuring the amount of antibody on the solid phase.
  • the antibody may be immobilized and the assay may involve measuring the amount of the amino acid sequence on the solid phase.
  • Amino acid sequences having the desired activity may be identified from their nucleic acid sequence.
  • the present invention relates to methods for producing proteins and identifying protein function, structure and/or activity.
  • the invention also provides for compositions, systems and apparatuses for accomplishing these methods.
  • preferred methods of the invention employ proteins produced by in vitro cell-free transcription and translation systems and are tolerant of high levels of impurities in the protein preparations.
  • the methods of the invention are especially conducive to the rapid, parallel, processing and/or screening of large numbers of proteins.
  • large numbers of proteins may be screened, preferably in parallel, for specific functional or structural attributes of interest.
  • attributes include, but are not limited to, the group consisting of: i) binding activity (i.e., the ability to participate in a binding interaction with a binding partner); ii) enzymatic activity; iii) chemical reactivity; iv) substrate activity (i.e., the ability to act as a substrate an enzyme or other catalyst); v) post-translational modification; and vi) regulatory activity (e.g., the ability to alter the regulation of a cell or other complex biological or biochemical system).
  • one embodiment of the invention comprises expressing a library of proteins (preferably, in a multi-well plate format) by in vitro expression of a library of nucleic acid constructs encoding the proteins; screening the library of proteins for proteins with an activity of interest and screening one or more proteins having the desired activity against a library of substances to identify modulators of the activity of these proteins.
  • the proteins are produced by in vitro cell-free translation methods, preferably by coupled transcription/translation methods. Applicants have demonstrated that these techniques are amenable to the parallel synthesis of large numbers of proteins in multi-well plates. Surprisingly, the proteins produced from these cell-free systems may be analyzed without purification, although in some applications it may be advantageous to purify the proteins prior to analysis, e.g., to remove interfering activities from the cell-free protein synthesis reagent. In especially preferred embodiments, the protein synthesis methods are designed to produce proteins that share a common chemical moiety (e.g., a detectable label) that allow the proteins to be purified, immobilized or detected using a uniform set of conditions.
  • a common chemical moiety e.g., a detectable label
  • the proteins can be screened for a variety of attributes of interest using conventional detection techniques for measuring protein attributes (e.g., techniques based on measurements of fluorescence, electrochemiluminescence, chemiluminescence, radioactivity, refractive index, magnetic fields, light scattering, etc.), preferably, using detection techniques that are carried out in a high-throughput and/or highly parallel fashion.
  • detection techniques for measuring protein attributes (e.g., techniques based on measurements of fluorescence, electrochemiluminescence, chemiluminescence, radioactivity, refractive index, magnetic fields, light scattering, etc.), preferably, using detection techniques that are carried out in a high-throughput and/or highly parallel fashion.
  • Activities of interest may be measured, e.g., via the measurement of the quantity of an analyte; the measurement of a property of a sample (e.g., temperature, luminescence, electrochemical activity, color, turbidity, etc.); the measurement of a chemical, biochemical and/or biological activity (e.g., an enzymatic activity); the measurement of a kinetic or thermodynamic parameter (e.g., the rate or equilibrium constant for a reaction), etc.
  • the assay techniques may be involve the detection of labels or, alternatively may not require labels (e.g., refractive index based techniques such as surface plasmon resonance).
  • the assay techniques may involve the detection of reactions of species in solution (homogenous assays) or may involve at least some species on a solid phase (solid phase or heterogeneous assays).
  • the screening assays are carried out in a parallel fashion in multi-well plates (e.g., 24, 96, 384, or 1536 well plates).
  • the assays are carried out using solid phase assay formats (e.g., assays that presence of the attribute of interest to the attachment or release of an assay component from a solid phase support).
  • the assays are solid phase assays employing ECL detection.
  • a plurality of attributes of a protein are measured in one well of a multi-well plate, e.g., by using a patterned array of assay domains on a surface of the well, the different domains designed for measuring different attributes.
  • the association or dissociation of labels from a surface can be measured in a washed format or, if the detection technique is surface-selective, in an unwashed format.
  • the surface e.g., the working electrode surface of a well in an ECL multi-well plate
  • a solution e.g., with a solution containing an ECL coreactant, such as TPA that provides an appropriate environment for the induction of ECL from ECL labels
  • the wash step may be omitted and, if appropriate, reagents such as ECL coreactants are added without first removing unbound labeled reagents ("unwashed" assay format).
  • reagents such as ECL coreactants
  • the surface selectivity af ECL measurements, especially in measurements involving the use of electrodes as solid phase assay supports, allows ECL measurements to be carried out in washed or unwashed formats.
  • unwashed heterogeneous assays may be carried out using other surface selective techniques such as surface proximity assay. In washed ECL assays, it is preferred that the ECL measurement be conducted within a short time period after the addition of the coreactant solution to avoid loss of signal due to dissociation of the reagent in a binding interaction.
  • the timing is less important because free ligand remains in solution and the effect of the addition of the ECL coreactant on the binding equilibrium is small.
  • the ECL measurements may be conducted as long as 1 hr after addition of the ECL coreactant solution with only small changes in signal.
  • the proteins to be analyzed are produced in a labeled form using an in vitro translation, e.g., by including tRNAs charged with labeled amino acids in the translation reaction. These proteins may be used either directly or after affinity purification using the attached label. These proteins are then either immobilized via the label or are detected via the label.
  • the labeled protein is contacted with a capture reagent that is immobilized on a solid phase, the capture reagent binding to the label so as to immobilize the labeled protein on the solid phase. Attributes of the protein may then be measured by solid phase assay.
  • the proteins can be analyzed for specific structural features or post-translational modifications by contacting them with binding reagents (e.g., antibodies) that are specific for those structural features or post-translational modifications and measuring the formation of binding complexes (preferably, via the detection of a second label attached to the binding reagent),
  • binding reagents e.g., antibodies
  • binding activity is measured by contacting the proteins with potential binding partners (e.g., specific proteins, peptides, lipids, drugs, nucleic acids, sugars and oligosaccharides, etc.) and measuring the formation of binding complexes (preferably, via the detection of a second label attached to the potential binding partner).
  • potential binding partners e.g., specific proteins, peptides, lipids, drugs, nucleic acids, sugars and oligosaccharides, etc.
  • the proteins are analyzed for substrate activity by contacting them with an enzyme or other chemical modification reagent and measuring the modification of the protein, e.g., via a, preferably labeled, binding reagent specific for the modified or unmodified form (or, alternatively, by measuring the cleavage from or attachment to the protein of a labeled assay component.
  • the enzymatic activity e.g., kinase, phosphatase, protease, cyclase, lipase, nuclease, ligase, polymerase or glycosidase activity
  • the enzymatic activity e.g., kinase, phosphatase, protease, cyclase, lipase, nuclease, ligase, polymerase or glycosidase activity
  • the enzymatic activity e.g., kinase, phosphatase, protease, cyclase, lipase, nuclease, ligase, polymerase or glycosidase activity
  • Analogous methods exist for analyzing proteins that are not directly immobilized via the label.
  • the binding partner or binding reagent as described above, are preferably immobilized on a solid phase.
  • the binding of the labeled test protein to the immobilized reagent is, preferably, detected via the detection of the label.
  • Post-translational modification refers to structural modifications of proteins after they have been translated. These can be in the form of covalent modifications or non-covalent.
  • Examples of covalent post-translational modifications that can be detected according to the invention include signal peptide cleavage, phosphorylation, acetylation, adenylation, proteolysis, amino peptidase clipping, arginylation, disulphide bond formation and cleavage, amidation, glycosylation, isoprenylation, myristoylation, ubiquitination, SUMOlation, and covalent addition of proteins including Agp 12 and Nedd8.
  • non-covalent post-translational modifications examples include changes in the structure or folding state of a given protein (e.g., denaturation) or its non-covalent interactions with other proteins, nucleic acid, carbohydrate, drugs, compounds or lipids.
  • changes in the structure or folding state of a given protein e.g., denaturation
  • other proteins e.g., nucleic acid, carbohydrate, drugs, compounds or lipids.
  • prions structure from PrPc to PrPsc resulting in resistance to protease activity.
  • Other examples include the binding of a protein as a homo or hetero polymer, insertion in to a lipid membrane, or binding to a glycosyl group, or binding to a drug or compound.
  • drug or compound binding include for example the interaction of steroid receptors with a steroid.
  • In vitro translation systems may introduce post-translational modifications into translated proteins. Often, these post-translational modifications (especially as introduced using eukaryotic translation systems, in particular, the reticulyte systems) may indicative of post-translational modifications that are observed in vivo .
  • the methods of the invention may be used to identify proteins that receive specific types of post-translational modifications.
  • the post-translational modifications may be the same as the modifications which are introduced by, e.g., an enzyme of interest and may therefore interfere with the assay.
  • the protein is immobilized to simplify removal of the demodifying activity (e.g., via wash steps) prior to conduct of the assay.
  • an assay of the invention for a characteristic of protein may comprise the step of treating the protein with a modifying activity (this treatment is most preferably applied after immobilization of the protein on a solid phase) to uncover a nascent activity of the protein.
  • a modifying activity this treatment is most preferably applied after immobilization of the protein on a solid phase
  • Especially preferred treatments are protease treatment, phosphorylation and dephosphorylation.
  • This technique enables the screening of proteins in different modification states and analysis of the effect of modifications on activity.
  • the protein is immobilized to simplify removal of the modification activity (e.g., via wash steps) prior to conduct of the assay.
  • the protein is first treated with demodifying activities then treated with modifying activities in order to ensure that the protein is in a defined, reproducible modification state.
  • the invention relates, in part, to the production and analysis of proteins formed by the in vitro transcription and translation of nucleic acid constructs.
  • These nucleic acid constructs are characterized by their ability to direct an RNA polymerase to produce an RNA transcript that, in turn, is able to direct the synthesis of a protein or polypeptide sequence.
  • the nucleic acid construct may comprise DNA and/or RNA (preferably, DNA) and may include regions that are single stranded, double stranded (through base pair binding to its complementary sequence), or partially double stranded.
  • the nucleic acid constructs preferably, contain an RNA polymerase promoter sequence that directs the activity of a RNA polymerase to copy at least a portion of the nucleic acid construct to produce an RNA transcript.
  • This transcript preferably, either encodes a protein or polypeptide or a portion of a protein or polypeptide.
  • the proteins or polypeptides may correspond to amino acid sequences that are found in nature or may be man-made sequences (i.e., sequences not found in nature).
  • the nucleic acid constructs may include synthetic nucleic acid analogs or derivatives that are capable of being copied by transcriptional machinery.
  • Preferred nucleic acid constructs include plasmid based cloning vectors containing a cloned DNA sequence of a protein or amino acid sequence (natural or man-made). These plasmid based vectors are, preferably, produced by growing the plasmids in E.coli cultures and purifying the plasmid DNA to obtain the DNA in a form that can be used. Alternate preferred nucleic acid constructs include DNA sequences that have been generated using PCR or other amplification protocols.
  • the production of nucleic acid constructs by nucleic acid amplification avoids the use of cloning vectors and cloning protocols that involve transfer and amplification of the DNA in a host organism.
  • DNA and or RNA from an organism is amplified and engineered during the PCR to generate a nucleic acid construct of the invention that is able to direct the production of a RNA sequence under direction of a RNA polymerase. This RNA sequence is then able to produce the protein or proteins of interest via the translation of the RNA sequence.
  • Preferred cloning vectors used in the nucleic acid constructs of this invention include those that contain RNA polymerase sites positioned such that they will allow the production of an RNA strand from the cloned DNA.
  • Suitable cloning vectors containing RNA polymerase sites are available from a number of commercial suppliers including Promega (Madison, WI) Invitrogen (Carlsbad, CA) Novagen (Madison, WI) Clontech (Palo Alto CA).
  • Examples of the RNA polymerases are SP6, T7 and T3.
  • Examples of cloning vectors that contain promoters for these polymerases include pSP72 (Promega, Madison WI), pCITE-2 and -4 (Novagen, Madison, WI).
  • cloning in to these vectors are well known in the art and reagents and kits are commercially available (Invitrogen, Carlsbad, CA. Clontech, Palo Alto, CA). Many clones are available in suitable vectors; especially preferred nucleic acid constructs include clones distributed by the I.M.A.G.E. Consortium (Lawrence Livermore National Lab, Livermore, CA).
  • gene sequences of interest in vectors are correctly orientated relative to the RNA polymerase sites and within a reasonable distance such as from 0 to 2000 bases, ideally 0 to 500, most ideally 0 to 200.
  • the methods for directionally cloning are well known in the art and are typically part of most cloning methods for mRNA.
  • the DNA sequences are cloned without directionality which results in a mixture of clones with the DNA sequence in both of the possible orientations relative to the RNA polymerases site.
  • Suitable vectors for use with the invention are vectors that contain additional sequences to the previously described vectors which allow the cloning of the gene of interest in such a way that these additional sequences are incorporated into the transcribed RNA and thus into the translation products of these RNA's.
  • This approach has been used for the cloning of many genes to allow the production of translation products that are fused to other sequences of amino acids that allows the translations products to be detected and or purified.
  • These additional sequences of amino acids are typically referred to as affinity tags, epitope tags, or purification tags.
  • vectors examples include pGADT7, pGBKT7 (Clontech, Palo Alto CA) pcDNA5/FRT/V5-His-TOPO, pSecTag/FRT/V5-His-TOPO (Invitrogen, Carlsbad, CA).
  • affinity, epitope or purification tags include, V5, myc, His, FLAG, GST, and MBP.
  • Nucleic acid constructs can be replicated using standard nucleic acid replication methods including growth in culture (e.g., using standard methods for replicating plasmid DNA) and in vitro amplification methods such as PCR, NASBA ( Romano JW, et al., Immunol Invest. 1997;26:15-28 ), rolling circle replication ( Zhang DY, et al., Mol Diagn. 2001, 6:141-50 ). These steps are, preferably, followed by purification of the constructs. Methods for the isolation of plasmid DNA are well known in the art; examples of commercially available kits are available from Promega (Madison WI) and Qiagen (Los Angeles, CA).
  • nucleic acid constructs of the invention obviates the cloning steps and makes use of in vitro methods throughout. With these methods the sequences of interest are amplified using standard nucleic acid amplification methods such as RTPCR, PCR, NASBA and rolling circle amplification.
  • oligonucleotide primers are selected such that the amplification product includes the gene sequence of interest as well as other additional features such as an RNA polymerase recognition site,
  • other additional features are engineered into the amplification product such as an ATG initiation codon (preferably, modified to match the Kozak sequence such as CCACCATGG to improve translation, see, e.g., Kozak, M., J. Mol Biol. (1987) 196, 947 ), a termination of translation site, a 3' UTR and/or a poly A sequence.
  • a 5' internal ribosome entry site may also be introduced.
  • RNA, genomic DNA, cDNA, total RNA or cloned DNA examples of samples include RNA, genomic DNA, cDNA, total RNA or cloned DNA.
  • sources of the sequences of interest may be purified or unpurified.
  • purified DNA this typically would consist of a mixture of sequences that have been purified from the tissue or cell sample.
  • unpurified material this could take the form of whole cells, tissue samples, cell lysates and tissue homogenates. Examples of methods suitable for use in the in vitro production of nucleic acid constructs of the invention are illustrated in the following publications ( Bateman, JF. et al, Human Mutation, (1999) 13, 311 . Kirshgesser, M. et al, (1998) 36, 567. Rao, VR., et al, (1999) J. Biol. Chem. 274, 37893 ).
  • the nucleic acid constructs of the invention may be RNA generated using in vitro methods or via the use of viral RNA replication as found in Picornaviruses, Myxovirus, Reovirus and RNA bacterophage R17, Qb, MS2 and 12; using RNA dependent RNA polymerases, These RNA viruses can be used to clone and isolate specific genes using standard recombinant methods. Alternatively these RNA dependent RNA polymerases may direct the in vitro production of RNA for translation from an existing RNA template.
  • the RNA template may be produced from a DNA clone or DNA sequence or from an in vitro constructed RNA template.
  • RNA is treated with calf intestinal phosphatase, to remove the 5' phosphate from partial transcripts, these treated RNAs are then treated with tobacco acid pyrophosphatase to remove the 5' RNA cap, exposing the 5' phosphate.
  • the modified mRNA is then ligated to a sequence effective in directing the activity of a RNA dependent RNA polymerase. These ligated and modified mRNA sequences are then hybridized to an RNA oligonucleotide that hybridizes to the 3' poly A and introduces a site for RNA dependent RNA polymerase.
  • RNA sequences are then made double stranded using a RNA dependent RNA polymerase followed by the synthesis of RNA for translation using a RNA dependent RNA polymerase that is activated by the sequence attached at the 5' end of the mRNA.
  • a RNA dependent RNA polymerase that is activated by the sequence attached at the 5' end of the mRNA.
  • the addition of the 5' sequence for the RNA dependent RNA polymerase may be carried out with out the use of the calf intestinal phosphatase and tobacco acid pyrophosphatase steps that ensure that only full-length RNA sequences are subjected to the production of RNA for translation using the RNA dependent RNA polymerase.
  • translation and, optionally, transcription of nucleic acid constructs to produce proteins is carried out in an assay plate used for carrying out an assay for an activity of interest (e.g., plates as described elsewhere in this specification having assay domains coated with i) a substrate of an activity, ii) a binding reagent specific for a substrate of an activity, iii) a binding reagent specific for a product of an activity, iv) a binding reagent specific for a post-translational modification of the translation products, v) a binding reagent suitable for immobilizing the translation products, etc.).
  • the assays of the invention are then carried out in this same plate.
  • the translation reaction is carried out in one container, preferably, the well of a multi-well plate and the translation products transferred to an assay plate for analysis. This embodiment may be advantageous when only small amounts of translation products are used in an assay.
  • binding species as used herein, is used to describe a molecular species that is able to bind to another molecular species, its binding partner. These binding interactions are characterized in that they are non covalent, or covalent having dissociation constants which are, preferably, lower than 1 mM, more preferably lower than 100 ⁇ M, more preferably lower than 10 ⁇ M and most preferably higher than 1 ⁇ M.
  • binding species and binding partners include biotin, antibodies, streptavidin, avidin, EDTA, chelates (e.g., EDTA, NTA, IDA, etc.), antigens, fluorescein, haptens (e.g., fluorescein, digoxigenin, etc.), proteins, peptides, drugs, nucleic acids, nucleic acid analogues, lipids, carboyhydrates, protein A, protein G, protein L, receptors, ligands, inhibitors, lectins, enzymes, substrates, transition state analogues, mechanism based inhibitors, epitopes, affinity tags (e.g., epitope tags such as his(6), glutathione, Myc, S-tag, T7-Tag), etc.
  • affinity tags e.g., epitope tags such as his(6), glutathione, Myc, S-tag, T7-Tag
  • the assays of the invention may utilize labels.
  • Babel or detectable label is used herein is used to describe a substance used to detect (directly or indirectly) a molecular species.
  • the label may be the molecular species itself or it may be linked to the molecular species.
  • labels are used in order to follow or track a given molecular species, for example, to determined its distribution and concentration (as, for example, a radio-labeled drug molecule is used to determine its pharmacological properties when introduced into an animal or patient).
  • a label is introduced into a binding species so as to allow the binding species to be used to track and/or determine the presence and/or amount of a binding partner of the binding species.
  • immunoassays using labeled antibodies can be used to detect and determine binding partners (analytes) bound by the antibody.
  • binding partners analytes bound by the antibody. Examples of this approach are exemplified by the ECL-based clinical immunoassays sold by Roche Diagnostics (Indianapolis, IN) under the Elecsys tradename.
  • labels are used which may be detected directly, e.g., on the basis of a physical or chemical property of the label (e.g., optical absorbance, fluorescence, phosphorescence, chemiluminescence, electrochemiluminescence, refractive index, light scattering, radioactivity, magnetism, catalytic activity, chemical reactivity, etc.).
  • directly detectable labels include, radioactive labels, fluorescent labels, luminescent labels, enzyme labels, chemiluminescent labels, electrochemiluminescent labels, phosphorescent labels, light scattering or adsorbing particles (e.g., metal particles, gold colloids, silver colloids), magnetic labels, etc.
  • labels are used which may be detected indirectly via interactions with species comprising directly detectable labels.
  • These indirectly detectable labels are, preferably, binding species; these binding species readily allow the binding to a binding partner that is labeled with a directly detectable label.
  • indirectly detectable labels include binding species as described above, e.g., antibodies, antigens, haptens, avidin, biotin, streptavidin, flourescein, nucleic acid sequences, nucleic acid analogue sequences, epitope tags (such as myc, FLAG, GST, MBP, V5), digoxigenin, etc.
  • Homologous when used herein is used to describe a molecular species' relationship with another molecular species.
  • Two molecular species are homologous when they share a set of molecular properties such as composition, function or structure.
  • the extent of homology can be described in terms of the degree of sequence similarity.
  • two proteins or nucleic acids may be considered homologous when they have greater than 20% sequence in common, ideally greater than 30%, or preferentially more than 40% sequence in common.
  • Homology can also be described for protein and nucleic acid sequences based on the data from a BLAST search of the sequences in the NIH genebank database (http://www.ncbi.nlm.nih.gov/BLAST).
  • an E value of 1 assigned to a hit from a homology search can be interpreted as meaning that in a database of the current size one might expect to see 1 match with a similar score simply by chance. Thus any E value less than 1 would not be expected to happen by chance and would indicate that the two sequences are homologous.
  • This approach to description of homology can also be formally applied to other molecular characteristics such as structure as quantified using VAST or a similar protein structure searching algorithm ( http://ww.ncbi.nlm.nih.gov/Structure/VAST/vast.shtml ).
  • the VAST p-value is a measure of the significance of the comparison, expressed as a probability. For example, if the p-value is 0.001 then the odds are 1000 to 1 against seeing a match of this quality by pure chance. Thus a VAST p-value of less than 0.001 demonstrates a significant degree of homology.
  • the invention relates, in part, to the production of proteins in cell free systems and the analysis of these proteins.
  • Protein synthesis is, preferably, accomplished using cell lysates or extracts (crude or partially purified) that contain the machinery necessary for protein synthesis. This machinery is found in most living cells, however, certain cell types are preferred because of their high protein synthesis activity.
  • Three preferred translation systems for producing proteins are the bacterial (more preferably E coli extract, most preferably E coli S30 cell free extract), plant germ (most preferably, wheat germ) and reticulocyte (most preferably, rabbit reticulocyte) lysate translation systems.
  • the cell lysates are supplemented with additional components such as ATP, tRNA, amino acids, RNA polymerases, microsomes, protease inhibitors, proteosome inhibitors, etc. that enhance the functioning of the translation machinery, provide a missing component of the machinery, inhibit protein degradation or provide an additional activity such as transcription or post-translational modification.
  • the cell-free system is reconstituted from individual purified or partially purified components (e.g., a reconstituted E coli translation system using recombinant components as described in Shimizu et al. (2001) Nature Biotechnology 19, 751-755 ).
  • a cell-based translation system is used.
  • the E.coli systems are advantageous when a gene has been cloned into a vector with prokaryotic regulatory sequences, such as the promoter and ribosome binding site. These systems are coupled in that transcription and translation can occur simultaneously.
  • the E.coli extract systems have been the subject of much optimization and allow the production of mg amounts of protein using large scale (1ml) reactions fed with reagents through semi-permeable membranes.
  • Wheat germ and the rabbit reticulocyte based cell free systems are suitable for the translation of mRNA into proteins.
  • these lysates are supplemented with RNA polymerases so that they carry out both transcription, to generate the mRNA, and translation to produce protein thus producing what is called a coupled system as seen in E.coli.
  • the lysates based on the wheat germ and the rabbit reticulocyte may also be supplemented with other components in order to introduce additional activities to these lysates.
  • a lysate is supplemented with microsomes (preferably, dog pancreatic microsome preparations) or xenopus oocyte extract to allow for certain post-translation modifications as well as the processing of proteins which are secreted, inserted or associated with membranes.
  • proteins are produced using a cell free translation system that is supplemented with tRNAs that are charged with labeled or amino acids so that the proteins incorporate the label. It is preferable to use a tRNA that corresponds to an amino acid with a hydrophilic or charged side chain (most preferably tRNA lys ) so as to make it more likely that the label will be found on the exterior of the protein.
  • tRNA lys a variety of suitable charged tRNAs are commercially available including tRNA lys charged with lysines that are labeled on their side chains with fluorophores (such as fluorescein and BODIPY) or binding species (such as biotin).
  • tRNA lys a fraction of a specific tRNA (e.g., tRNA lys ) in a translation reaction (preferably, less than 50 %, more preferably, ⁇ 10%) is charged with a label so as to ensure that the number of incorporated labels in a protein molecule is low and that the labeling does not significantly influence the structure or functional properties of the protein.
  • solid phase supports are used for purifying, immobilizing, or for carrying out solid phase activity assays for analyzing the activity of one or more expressed proteins.
  • solid phases suitable for carrying out the methods of the invention include beads, particles, colloids, single surfaces, tubes, multi-well plates, microtitre plates, slides, membranes, gels and electrodes.
  • the solid phase is a particulate material it is, preferably, distributed in the wells of multi-well plates to allow for parallel processing of the solid phase supports.
  • Proteins of interest or other assay reagents may be immobilized on the solid phase supports, e.g., by non-specific adsorption, covalent attachment or specific capture using an immobilized capture reagent that binds, preferably specifically, the protein or assay reagent of interest. Immobilization may be accomplished by using proteins or assay reagents that are labeled with binding species that form binding pairs with immobilized capture reagents.
  • a protein is immobilized on a solid phase, the solid phase is washed and the protein is analyzed; the wash step allows for the rapid purification of the protein from other, potentially interfering, components of the translation reaction.
  • a protein is treated, prior to analysis, to add or remove post-translational modifications.
  • Especially preferred solid phase supports are electrode surfaces integrated into the wells of multi-well plates. Such devices allow ECL measurements to be carried out in a high-throughput, highly parallel, fashion.
  • Exemplary multi-well plates are disclosed in copending Provisional Application No. 60/301,932 entitled “Assay Plates, Reader Systems and Methods for Luminescence Test Measurements", filed on June 29, 2001 (particularly in the description of Plate Type B, Type C, type D, and Type E in Example 6.1) and U.S. Application Serial Nos. 10/185,274 and 10/185,363, filed June 28,2002 , each hereby incorporated by reference.
  • the electrodes in such plates preferably, comprise a carbon composite electrode material such a carbon ink.
  • the multi-well assay plate can incorporate the electrode in one or more wells of the plate.
  • the assay region e.g., a given well of a multi-well plate
  • at least one electrode in an assay region is suitable for use as a working electrode in an electrode induced luminescence assay
  • at least one electrode is suitable for use as counter electrode in an electrode induced luminescence assay.
  • the surface of the working electrode in an electrode induced luminescence assay can be used for immobilization of one or more assay components.
  • Electrodes used in the multi-well assay plates of the invention are typically non-porous, however, in some applications it is advantageous to use porous electrodes (e.g., mats of carbon fibers or fibrils, sintered metals, and metals films deposited on filtration membranes, papers or other porous substrates. These applications include those that employ filtration of solutions through the electrode so as to: i) increase mass transport to the electrode surface (e.g., to increase the kinetics of binding of molecules in solution to molecules on the electrode surface); ii) capture particles on the electrode surface; and/or iii) remove liquid from the well.
  • porous electrodes e.g., mats of carbon fibers or fibrils, sintered metals, and metals films deposited on filtration membranes, papers or other porous substrates.
  • the wells of multiwell plate can further comprise a plurality (e.g., 2 or more, 4 or more, 7 or more, 25 or more, 64 or more, 100 or more, etc.) of discrete assay domains.
  • Multi-domain multiwell plates which are adapted to allow assay measurements to be conducted using electrode induced luminescence measurements (most preferably, electrochemiluminescence measurements) are described in copending Provisional Application No. 60/318,293 entitled "Methods and Apparatus for Conducting Multiple Measurements on a Sample", filed on September 10, 2001, and U.S. Application Serial No. (Attorney Ref. No.
  • Multiple assay domains patterned on a surface of a well may be defined by physical boundaries which can include ledges or depressions in the surface, patterned materials deposited or printed on the surface, and or interfaces between regions of the surface that vary in a physical property (e.g., urettability). Such physical boundaries simplify the patterning of reagents on surfaces of a well by confining and preventing the spreading of small drops of reagents applied to an assay domain.
  • Multi-domain multi-well (MDMW) Plates provide a variety of advantages over conventional multi-well plates that only have one assay domain per well. For example, a MDMW Plate having N wells and M assay domains per well allows a panel of M assays to be run on a plurality of N samples. Conducting the same series of assays on conventional N-well plates would require M plates, M times more sample and reagents, and considerably more pipetting and plate handling steps to achieve the same performance. Conducting the same series of assays on conventional array "chips" would involve the handling and movement ofN chips and would likely not be compatible with standard plate handling equipment designed for use with multi-well plates.
  • Multi-well and Multi-domain Multi-well plates can be used in a plurality of diverse assays.
  • the same analyte is measured at different assay domains within a well, the different assay domains being designed to measure a different property or activity of the analyte.
  • an enzyme with multiple different activities is measured in a well comprising different assay domains that differ in their selectivity for each enzymatic activity of the enzyme (e.g., assay domain that comprise substrates for selected enzymatic activities and/or assay domains that are capable of capturing and measuring the substrates or products of selected enzymatic activity), that are designed to measure binding activities of the enzyme (e.g., assay domains comprising potential binding partners of the enzyme or that are designed to capture the enzyme so as to allow the measurement of interaction with potential binding partners in solution) and/or assay domains designed to measure the ability of the enzyme to act as a substrate for a second enzyme (e.g., a binding domains designed to allow for a specific binding assay of the product of the action of the second enzyme on the first enzyme).
  • assay domains that differ in their selectivity for each enzymatic activity of the enzyme
  • binding activities of the enzyme e.g., assay domains comprising potential binding partners of the enzyme or that are designed to capture the enzyme so as to
  • a well comprises a domain for measuring the amount of an enzyme (e.g., via a binding assay such as an immunoassay) and one or more other domains for measuring one or more activities associated with the enzyme; this allows the measured activity to be referenced to the amount of enzyme.
  • the inclusion of assay domains capable of capturing an enzyme of interest has the added advantage of allowing the purification of the enzyme from a crude sample within the assay well and/or the pretreatment of the enzyme with a modifying activity that exposes a nascent activity of the enzyme.
  • a well may also comprise an assay domain capable of capturing an enzyme of interest and one or more additional assay domains for measuring an activity of the enzyme of interest. Methods using such a well may include a wash step for purifying the enzyme from impurities in a crude enzyme preparation.
  • a series of nucleic acid constructs is obtained with the genes of interest located within the construct such that the gene of interest can be transcribed into RNA that can direct the synthesis of the protein encoded by the genes of interest.
  • the nucleic acid from these is then subjected to an in vitro transcription reaction and followed by an in vitro translation reaction. These two reactions can also be combined in a single in vitro reaction.
  • the protein produced in the transcription and translation reaction is also labeled using a modified tRNA that directs the incorporation of a modified amino acid during this step.
  • the proteins produced in the mix may be captured on to a solid phase.
  • solid phases suitable for carrying out the methods of the invention include beads, particles, colloids, single surfaces, tubes, multiwell plates, microtitre plates, slides, membranes, gels and electrodes.
  • the capture of the proteins is achieved using a binding partner specific for the modification introduced by the modified tRNA during the translation reaction.
  • a good example of how this is achieved is by the use of the biotin-lys-tRNA that results in a protein containing lysine residues modified with biotin.
  • the biotinylated proteins produced in this way are then captured on to a solid phase using an avidin or streptavidin coated solid phase.
  • Alternative methods for capture of the proteins produced include non-specific or passive adsorption, and via the binding to specific binding partners other than those for the modified amino acids introduced by the addition of modified tRNA (e.g., binding partners of affinity tags present in the expressed protein).
  • the proteins produced may be generated without the use of the modified tRNA and captured using alternative methods for capture, including non-specific or passive adsorption, and via the binding to specific binding partners for the produced proteins (e.g., binding partners of affinity tags present in the expressed proteins).
  • solid phase bound protein products of an in vitro transcription and translation reaction are produced, In the preferred embodiment these step are all carried out in a multiwell plate with 20 wells or more, or ideally 40 wells or more, or most ideally 84 wells or more that allows for the rapid and efficient analysis of the immobilized proteins.
  • a plurality of proteins preferably greater than 80, more preferably greater than 1000, more preferably greater than 10,000 and most preferably greater than 20,000
  • are produced in parallel and immobilized in separate wells of one or more multi-well plates e.g., 24, 96, 84 or 1536 well plates).
  • the protein products of the in vitro transcription and translation reaction are immobilized on to one of the following in a multi-well format: carbon containing surfaces or magnetic beads.
  • the protein products of the in vitro transcription and translation reaction are immobilized on to one of the plates described in more detail in copending Provisional Application No. 60/301,932 (entitled “Assay Plates, Reader Systems and Methods for Luminescence Test Measurements", filed on June 29,2001, and U.S. Application Serial Nos. 10/185,274 and 10/185,363, filed June 28, 2002 , each hereby incorporated by reference, and particularly in the description of Plate Type B, Type C, Type D, and Type E in Example 6.1. Immobilization in these multi-well plates may be advantageous, even in applications that do not involve ECL measurements, do to the excellent adsorption properties and high binding capacity of the carbon-containing electrode surfaces.
  • a protein expressed by the expression methods of the invention is characterized by its ability to bind to a potential binding partner (e.g., a biologically relevant binding partner or a binding reagent that specifically binds a structural characteristic such as an epitope or post-translational modification).
  • a potential binding partner e.g., a biologically relevant binding partner or a binding reagent that specifically binds a structural characteristic such as an epitope or post-translational modification.
  • the binding event is measured using technique that does not require a detectable label (e.g., surface plasmon resonance, agglutination).
  • the binding event is measured using a technique the measures a change in a property of labels linked to the protein and/or potential binding partner (e.g., techniques that measure changes in fluorescence polarization, lifetime or energy transfer).
  • one of the protein or potential binding partner is immobilized on a solid phase support so that the binding event may be measured by measuring the accumulation of the other member of the binding pair on the solid phase. Most preferably, this measurement is accomplished via a detectable label attached to the other member of the binding pair. In such assay methods, the number of binding interactions is correlated to the accumulation of labels on the solid phase; this accumulation of labels being measurable by a variety of techniques (e.g., fluorescence for fluorescence labels, enzyme activity for enzyme labels, ECL for ECL labels, etc.). Most preferably, the solid phase is an electrode material, the labels are ECL labels and the accumulation of labels is measured via an ECL measurement.
  • a protein expressed by the expression methods of the invention is characterized by its catalytic activity or its ability to act as a substrate in the presence of a catalytic activity or other chemical activity.
  • activity is characterized as a joining (i.e., an activity that joins two or more species, e.g., nucleic acid ligase activity, nucleic acid polymerase activity, protein translation activity, ubiquitin ligase activity, etc.) and/or cleaving activity (i.e., an activity that results in the cleaving of a species into two or more components, e.g., protease activity, nuclease activity, glycosidase activity, etc.), the activity can be measured using techniques analogous to those described for binding reactions.
  • an activity that joins a first substrate having a first label with a second substrate is measured by immobilizing the second substrate on a solid phase support (e.g., by passive adsorption or by capture via the specific binding of the second substrate or a second label on the second substrate to a binding reagent on the solid phase support) and measuring the amount of the first label on the solid phase.
  • a cleaving activity is measured by using a substrate having a first label that is immobilized on a solid phase support (e.g., by passive adsorption or by capture via the specific binding of a second label on the substrate to a binding reagent on the solid phase support) and measuring the loss of labels from the solid phase in the presence of the activity.
  • an activity is measured by using a substrate that exhibits a detectable change in a chemical or physical property when acted on by the activity of interest (e.g., luminescence, color, ability to act as an ELC coreactant, etc.).
  • a detectable change in a chemical or physical property when acted on by the activity of interest (e.g., luminescence, color, ability to act as an ELC coreactant, etc.).
  • a nucleic acid construct is preferably obtained with the genes of interest located within the construct such that the gene of interest can be transcribed into RNA that can direct the synthesis of the protein encoded by the genes of interest.
  • the nucleic acid construct is then subjected to an in vitro transcription reaction and followed by an in vitro translation reaction. These two reactions can also be combined in a single in vitro reaction.
  • the protein product (or, alternatively, protein obtained by any other method) is than analyzed for the presence of post-translational modifications of interest.
  • a plurality of constructs comprising different genes are prepared and the protein products of these genes produced and analyzed, most preferably in parallel in the wells of multiwell plates.
  • the protein produced is also labeled, most preferably by using a modified tRNA that directs the incorporation of a modified amino acid during this step.
  • the reaction mix is then subjected to assays for protein function, and or activity and or structure.
  • the protein in the mix is captured on to a solid phase using a binding protein specific for the label introduced during the translation reaction.
  • a good example of how this is achieved is by the use of the biotin-lys-tRNA that results in a protein containing lysine residues modified with biotin.
  • the biotinylated proteins produced in this way are then captured on to a solid phase using an avidin coated solid phase.
  • solid phase examples include beads, particles, colloids, single surfaces, tubes, multi-well plates, microtitre plates, slides, membranes, gels and electrodes,
  • the preferred plates are described in more detail in copending Provisional Application No. 60/301,932 (entitled “Assay Plates, Reader Systems and Methods for Luminescence Test Measurements", filed on June 29, 2001, hereby incorporated by reference) and particularly in the description of Plate Type B, Type C, Type D, and Type E in Example 6.1 and U.S. Application Serial Nos. 10/185,274 and 10/185,363, filed June 28, 2002 , each hereby incorporated by reference.
  • proteins immobilized on to a solid phase the proteins are then available for an assay to determine if they have been subject to any structural modifications or post-translational modifications.
  • These post-translational modifications are then detected using a labeled binding species specific for the post-translational modification that binds to the immobilized proteins containing these post-translational modifications.
  • An example of how this is achieved is illustrated by phosphorylation of tyrosines.
  • This post-translational modification is carried out by protein kinases and this is readily detected using antibodies specific to phosphotyrosine. In this example the antibody would be labeled with a detectable label and the bound signal detected to determine the amount phosphorylation for a given protein form the translation reaction.
  • a reagent is Abzyme (IGEN International, Inc., Gaithersburg MD) that is an antibody to phosphotyrosine labeled with an electrochemiluminescent (ECL) label.
  • ECL electrochemiluminescent
  • post-translational modifications examples of post-translational modifications that can be detected with antibodies include ubiquitinylation, SUMO, other phosphorylated sequences, acetylation, prenylation, farnesylation, and geranylation.
  • a labeled substrate for a given post-translation modification is included in the translation reaction.
  • substrates include ubiquitin, SUMO-1, Nedd8 or Agp12 or homologous substrates.
  • labels that could be used include, metal chelates, biotin, binding species, digoxigenin, enzymes, fluorophores, luminescent species and ECL labels.
  • ubiquitin is labeled with an ECL label.
  • a labeled substrate for a given post-translational reaction results in the production of a labeled protein.
  • Proteins that are labeled in this way through the action of a post-translational modification and are also labeled as described using modified tRNA contain two, preferably different, labels. Thus these proteins are readily detected by immobilization through one of the labels and detected with the other.
  • the protein is modified with a biotin during translation with biotin-Lys-tRNA and subjected to post-translational modification using ECL labeled ubiquitin.
  • the invention involves either obtaining clones or the production of clones in a desired cloning vector or nucleic acid construct.
  • the DNA from these is then subjected to an in vitro transcription reaction and followed by an in vitro translation reaction. These two reactions can also be combined in a single in vitro reaction.
  • the protein produced is also labeled using a modified tRNA that directs the incorporation of a modified amino acid during this step.
  • the reaction mix is then subjected to assays for post-translational modification.
  • the protein in the mix is then captured on to a solid phase using a binding protein specific for the post-translational modification.
  • an antibody specific for a post-translational modification for example an antibody to ubiquitin immobilized on to a solid phase.
  • This allows the capture of proteins that have been ubiquitinated on to the solid phase.
  • the solid phase include beads, particles, colloids, single surfaces, tubes, multiwell plates, microtitre plates, slides, membranes, gels and electrodes.
  • biotin-lys-tRNA in the translation reaction resulting in a protein containing lysine residues modified with biotin.
  • streptavidin also including avidin or anti-biotin antibodies
  • antibodies are available to ubiquitinylation, SUMO, other phosphorylated sequences, acetylation, which would readily allow the capture of the proteins produced in an in vitro translation reaction that are subjected to these post-translational modifications.
  • clones are either preferably produced or obtained in the desired vectors or nucleic acid constructs.
  • the constructs are then subjected to an in vitro transcription reaction and followed by an in vitro translation reaction. These two reactions can also be combined in a single in vitro reaction.
  • the protein product, or alternatively a protein obtained by other means is then incubated under conditions conducive to the activity of interest (pH, temp, presence of cofactors, substrates, energy sources, etc.) analyzed for the presence of the activity.
  • a plurality of constructs comprising different genes are prepared and the protein products of these genes produced and analyzed, most preferably in parallel in the wells of multi-well plates.
  • Such screens can be used, for example, to identify proteins with a specific category of activity (e.g., all tyrosine kinases) or to elucidate biochemical pathways by identifying the enzyme responsible for a reaction of a known substrate.
  • a panel of proteins is screened against a panel of substrates (the substrates, preferably, being immobilized as an array) to identify previously unknown enzyme/substrate pairs.
  • the substrate array is immobilized within the wells of a multi-well plate so that a large number of proteins can be quickly screened in parallel.
  • the protein produced is also labeled, preferably by using a modified tRNA that directs the incorporation of a modified amino acid during this step.
  • the protein in the mix is captured on to a solid phase using a binding protein specific for the label introduced during the translation reaction.
  • a binding protein specific for the label introduced during the translation reaction is by the use of the biotin-lys-tRNA that results in a protein containing lysine residues modified with biotin.
  • the biotinylated proteins produced in this way are then captured on to a solid phase using a streptavidin coated solid phase. It is also possible to use other binding species specific for biotin including avidin and anti-biotin antibodies.
  • the immobilized proteins are, optionally purified of other components of the translation system (e.g., by washing the solid phase) and are then analyzed for their enzyme activity. This is typically determined by the addition of the desired substrate for the activity of interest.
  • the substrate is then acted on by the immobilized enzyme activity produced by the transcription and translation reactions to produce the product of the enzyme activity of interest.
  • the activity of the enzyme is then determined by the detection and or quantitation of the product of the reaction.
  • the loss of the substrate may also be determined.
  • the measurements are, preferably, carried out as described above by i) measuring a detectable change in a property of a substrate on reaction of the substrate to form product; ii) measuring the binding activity of a substrate or product for binding reagents specific for one of the product and substrate or iii) through a measurement of joining or cleaving activity.
  • the protein is treated with a post-translational modification activity prior to conducting the enzymatic assay so as to uncover a nascent activity/
  • preferred enzyme activities examples include kinases, hydrolases, protease, polymerases, glycosidases, and phosphatases.
  • Other preferred enzyme activities that can be measured include the activities of enzymes that catalyze the post-translational modification of proteins, e.g., signal peptide cleavage, phosphorylation, acetylation, adenylation, proteolysis, amino peptidase clipping, arginylation, disulphide bond formation and cleavage, amidation, glycosylation, isoprenylation, myristoylation, ubiquitination, SUMOlation, Agp12 ligation, Nedd8 ligation and covalent addition of proteins.
  • enzyme activities which may be measured include cleaving or joining enzymes such as amidases, proteases, peptidases, glycosidases, saccharases, glycopeptidases, nucleases (including ribonucleases and deoxyribonucleases), endonucleases (including restriction endonucleases), exonucleases, ribosomes, ribosomal RNA, ribozymes, self-splicing molecules such as introns or inteins, esterases, phosphodiesterases, phosphorylases, AP endonucleases, polymerases (e.g., DNA or RNA polymerases), nucleic acid repair proteins, amino peptidases, carboxy peptidases, aminoacyl-tRNA synthetases, ADP-ribosyl transferases, proteases of the complement pathway, proteases of the thrombolytic pathways, transferases, endoglycosidases,
  • glycosyl transferases or glycogen synthases ligases, ubiquitin-protein ligases, trans-glutaminases, integrases, and DNA glycosylases (e.g., uracil-DNA glycosylases), helicases, etc.
  • substrates examples include, poly (Glu-Tyr), peptides, proteins, and proteins produced by a process of transcription and translation.
  • the substrate is a generic substrate that is broadly targeted acted on by a genus of enzymes (by way of example, applicants have found that poly (Glu-Tyr) can be used as a generic substrate in screens for tyrosine kinases.
  • the product of the action of a tyrosine kinase on the poly (Glu-Tyr) is to generate poly (glu-Tyr) containing phosphorylated tyrosines which is readily detected in a sandwich binding assay format using an antibody to phosphotyrosine immobilized on to a solid phase and labeled anti phospho-tyrosine antibody such as Abzyme (IGEN International, Inc., Gaithersburg, MD).
  • the proteins produced in the translation reaction may also be subjected to the action of a post-translational modification prior to determination of its enzyme activity. Examples of this include the phosphorylation of a protein from the translation reaction prior to its assay for kinase activity.
  • the methods of the invention may be used for identifying post-translational modifications that regulate enzymatic activity.
  • the invention is directed to the identification of proteins with self-modifying activities.
  • a protein expressed according to the methods of the invention (and, preferably, immobilized as described above) is incubated under conditions conducive to the activity of interest.
  • the same protein is then assayed for the presence of a self-modification of interest (e.g., via its binding activity for a binding reagent specific for the modification of interest or via the cleavage or joining of a labeled component from or to the protein.
  • the protein is immobilized on a solid phase (as described above) and the extent of modification assays measured according to the solid phase assay techniques described above.
  • the invention is directed to efficient methods for screening proteins for their ability to modify both themselves and other species. Both these activities may be biologically relevant (particularly for receptor kinases) and also provide complementary indicators of a proteins function.
  • this class of methods will be illustrated through assays for the identification of the in vitro kinase activity and autophosphorylation activity of proteins.
  • the proteins are immobilized onto a surface of a solid phase such as the surfaces in wells of multi-well plates (according, e.g., to the immobilization methods described above).
  • the proteins are incubated under conditions appropriate for a modifying activity of interest (e.g., kinase activity) in the presence of a substrate for the activity of interest (preferably, a generic substrate such as poly(glu,tyr) in the case of tyrosine kinase activity).
  • a substrate for the activity of interest preferably, a generic substrate such as poly(glu,tyr) in the case of tyrosine kinase activity.
  • the presence of self-modified proteins on the solid phase is measured using the solid phase assay techniques of the invention (e.g., via the use of labeled anti-phosphotyrosine antibodies for measuring auto-phosporylation).
  • the presence of modified substrate is also measured via the assay techniques of the invention (e.g., by carrying out a sandwich immunoa
  • the measurement of the modified substrate can be carried out in a separate assay compartment (e.g., by transferring the kinase reaction supernatants to another multi-well plate.
  • both measurements can be carried out in the same well by including in the well two discreet assay domains: an assay domain for immobilizing the test protein (e.g. an assay domain having an immobilized capture reagent, such as avidin, for capturing a test protein comprising a binding species such as biotin) and a discreet assay domain having appropriate reagents for measuring the modified substrate (e.g., an assay domain comprising an antibody specific for the modified substrate such as an anti-phosphotyrosine antibody.
  • an assay domain for immobilizing the test protein e.g. an assay domain having an immobilized capture reagent, such as avidin, for capturing a test protein comprising a binding species such as biotin
  • a discreet assay domain having appropriate reagents for measuring the modified substrate e.g., an assay
  • the invention also provides for an in vitro assay to screen for candidate substances, e.g., small molecules, proteins, nucleic acids, lipids, etc. that are effective at inhibiting or activating the kinase activity (against self and/or other substrates) of the identified kinases.
  • candidate substances e.g., small molecules, proteins, nucleic acids, lipids, etc. that are effective at inhibiting or activating the kinase activity (against self and/or other substrates) of the identified kinases.
  • the invention includes in vitro assays for the auto-phosphorylating activity and/or substrate phosphorylating activity for potential kinases selected from the list of kinases below.
  • the auto-phosphorylation assays are conducted using kinases that are expressed and immobilized as described above.
  • the substrate phosphorylation assays employ poly(glu,tyr) as a generic substrate.
  • kinases for use with the assays of the invention are Ephb3, Ephb4 and Fgfr4.
  • clones are preferably either produced or obtained in the desired vectors or nucleic acid constructs.
  • the DNA from these is then subjected to an in vitro transcription reaction and followed by an in vitro translation reaction. These two reactions can also be combined in a single in vitro reaction.
  • the protein product, or alternatively a protein obtained by other means is then incubated in the presence of a modifying activity, e.g., an enzymatic activity, under conditions conducive to the activity (pH, temp, presence of cofactors, substrates, energy sources, etc.) and analyzed for the presence of modifications derived from the activity.
  • a modifying activity e.g., an enzymatic activity
  • a plurality of constructs comprising different genes are prepared and the protein products of these genes produced and analyzed, most preferably in parallel in the wells of multi-well plates.
  • each protein is immobilized in a separate well of a multi-well plate.
  • a plurality of proteins are immobilized in an array in a well of a multi-well plate (or on a protein "chip") so that a panel of activities (preferably, one per well or chip) can be screened in parallel against a panel of potential substrates (preferably, a plurality per well or chip).
  • the protein produced is also labeled, preferably by using a modified tRNA that directs the incorporation of a modified amino acid during this step.
  • the reaction mix is then subjected to assays to determine if it is a substrate for an enzyme.
  • the protein in the mix is captured on to a solid phase using a binding protein specific for the label introduced during the translation reaction.
  • a good example of how this is achieved is by the use of the biotin-lys-tRNA that results in a protein containing lysine residues modified with biotin.
  • the biotinylated proteins produced in this way are then captured on to a solid phase using an avidin coated solid phase.
  • the immobilized are, optionally, purified of other components of the translation system (e.g., by washing the solid phase) and then analyzed for their substrate activity.
  • the immobilized proteins are contacted with an enzyme followed by an assay to detect the modifications of the immobilized proteins by the enzyme activity (e.g., by contacting the protein with a, preferably labeled, binding reagent that is specific for the modification or, in the case of a joining activity, via the use of a labeled co-substrate that is joined to the protein as a result of the activity).
  • an assay for protease substrates involves the use of specific binding reagents or antibodies that recognize the protein as altered by the protease. This readily allows the detection of the action of the enzyme and determination of a substrate for the enzyme.
  • a protein kinase is contacted with the immobilized proteins from the translation reaction and the resulting phosphorylation detected using a specific antibody for said phosphorylation as described above for post-translational modifications.
  • a ubiquitination enzyme, or enzyme mixture is contacted with the immobilized proteins from the translation reaction and the resulting ubiquitination detected using a specific antibody for said ubiquitination.
  • an alternative approach would be to make use of ubiquitin labeled with a detectable species such as an ECL label.
  • the proteins that are produced in the translation reaction may be assayed for substrate activity prior to immobilization and detection.
  • the proteins produced in the translation reaction may be subjected, prior to exposure to the test activity, to a pretreatment step that removes post-translational modifications introduced in the translation step (in particular, modifications of the type introduced by the test activity).
  • a pretreatment step that removes post-translational modifications introduced in the translation step (in particular, modifications of the type introduced by the test activity).
  • phosphates on a test protein that were introduced during protein translation may interfere with an assay for kinase activity.
  • pretreatment steps include phosphatase treatment, deubiquitination, de-farnesylation, de-geranylation, de-glycosylation.
  • the proteins produced in the translation reaction may also be subjected, prior to the determination of its substrate activity, to the action of a post-translational modification activity.
  • This embodiment enables the screening of specific modifications on substrate activity. Examples of this approach include the phosphorylation of a protein from the translation reaction prior to its assay for ubiquitination.
  • clones are preferably either obtained or produced in a desired cloning vector or nucleic acid construct.
  • the DNA from these is then subjected to an in vitro transcription reaction and followed by an in vitro translation reaction. These two reactions can also be combined in a single in vitro reaction.
  • the protein product, or alternatively a protein obtained by other means is then incubated in the presence of a potential binding partner and analyzed for formation of a binding complex.
  • a plurality of constructs comprising different genes are prepared and the protein products of these genes produced and analyzed, most preferably in parallel in the wells of multi-well plates.
  • each protein is immobilized in a separate well of a multi-well plate.
  • the potential binding partner may be immobilized and the test protein is introduced in solution.
  • a plurality of proteins are immobilized in an array in a well of a multi-well plate (or on a protein "chip") so that a panel of potential binding partners (preferably, one per well or chip) can be screened in parallel against a panel of proteins (preferably, a plurality per well or chip).
  • the potential binding partners are the species immobilized in the array and the test proteins are introduced into individual wells.
  • the protein produced is also labeled, preferably by using a modified tRNA that directs the incorporation of a modified amino acid during this step.
  • the reaction mix is then subjected to assays for protein binding activity.
  • the protein in the mix is captured on to a solid phase using a binding protein specific for the label introduced during the translation reaction.
  • a good example of how this is achieved is by the use of the biotin-lys-tRNA that results in a biotinylated protein containing lysine residues modified with biotin.
  • the biotinylated proteins produced in this way are then captured on to a solid phase using an avidin, or streptavidin coated solid phase.
  • the proteins may be treated with a post-translational modifying activity prior to analysis so as to uncover a nascent binding activity.
  • the proteins are then available for an assay to determine the protein's binding activities. These binding activities are determined by measuring the binding of the protein to a binding partner that is, preferably, labeled.
  • the binding partner is added to the proteins captured on to the solid phase and incubated to allow binding. Following this step the degree of binding is determined based on a determination of the amount of binding partner captured on to the solid phase, e.g. via measurement of a label on the binding partner.
  • the potential binding partner is immobilized on a solid phase support and the potential binding partner is contacted with a labeled test protein generated from a translation reaction.
  • the formation of binding complexes is determined by measuring the amount of labeled test protein on the solid phase.
  • Preferred potential binding partners include proteins, peptides (e.g., recognition sequences from a protein), lipids, phosphoinositides, nucleic acids, hormones (including steroids), drugs, etc.
  • the potential binding partner is a peptide that has a modification that is the same as a post-translational modification.
  • a potential binding partner may be a short phosphorylated peptide, preferably a putative recognition sequence from a known protein, e.g., the following recognition site from IkBa (H2N-LKKERLLDDRHD(p)SGLD(p)SMKDEEYC-COOH) [SEQ ID No. 1].
  • the binding assays of the invention may be used to identify proteins that bind to this recognition sequence.
  • Potential binding partners for use in the binding assays of the invention are, preferably, labeled with a detectable label.
  • the label may be used to detect the occurrence of a binding interaction, in particular, when the translated test protein is immobilized on a solid phase.
  • the label may be used to capture the potential binding partner on a solid phase via the interaction of the label with a capture reagent that binds the label.
  • Peptidic potential binding partners are, preferably, labeled at a Cys residue, e.g., the IkBa peptide [SEQ ID No. 1] described above is preferably labeled at the C-terminal Cys residue with a label such as the ECL label ORI-TAG maleimide.
  • Other methods for immobilizing a potential binding partner onto a solid phase include conventional immobilization methods such as passive adsorption, covalent linkage to a chemically activated surface including NHS ester, epoxide, maleimide chemistries and cross-linking reagents.
  • Useful cross-linking reagents include cross-linking reagents that comprise one or more functional groups capable of reacting with components of a lipid/protein layer or an electrode surface (e.g., imidoesters, active esters such as NHS esters, maleimides, a-halocarbonyls, disulfides such as pyridyldithiols, carbodiimides, arylazides, amines, thiols, carboxylates, hydrazides, aldehydes or ketones, active carbamates, glyoxals, etc.).
  • functional groups capable of reacting with components of a lipid/protein layer or an electrode surface
  • cross-linking agents include homo- and hetero-bifunctional cross-linking agents such as those sold by Pierce Chemical Co. and/or described in the 1994 Pierce Catalog and Handbook (Pierce Chemical Co., Rockford, IL, 1994 ), the chapters relating to cross-linking agents hereby incorporated by reference.
  • small molecules or peptides these can be directly immobilized using the chemically activated surfaces as described previously.
  • the small molecules are first coupled using typical chemistries such as NHS ester, epoxide, cross-linking chemistries (see above), maleimide to a carrier typically a large molecule such as a protein that can then be immobilized using a simple passive adsorption methods.
  • chemistries such as NHS ester, epoxide, cross-linking chemistries (see above)
  • maleimide to a carrier typically a large molecule such as a protein that can then be immobilized using a simple passive adsorption methods.
  • An example of this is the coupling of Cys or SH containing peptides to maleimide activated BSA.
  • the small molecules are first coupled to a another binding species such as biotin or fluorescein, these conjugates are then immobilized using a binding species:binding partner interaction such as biotin:streptavidin, biotin:avidin, biotin:antibiotin, fluorescein:anti-fluorescein.
  • binding species:binding partner interaction such as biotin:streptavidin, biotin:avidin, biotin:antibiotin, fluorescein:anti-fluorescein.
  • binding species:binding partner interaction such as biotin:streptavidin, biotin:avidin, biotin:antibiotin, fluorescein:anti-fluorescein.
  • binding species:binding partner interaction such as biotin:streptavidin, biotin:avidin, biotin:antibiotin, fluorescein:anti-fluorescein.
  • examples of such small molecule conjugates include thyroxine-biotin and tri
  • clones are preferably either obtained or produced in a desired cloning vector or nucleic acid construct.
  • the DNA from these is then subjected to an in vitro transcription reaction followed by an in vitro translation reaction to produce a first library of translated proteins.
  • the transcription and translation reactions can be combined.
  • proteins are labeled with a first label during the translation reaction, preferably by introducing a modified tRNA that directs the incorporation of a modified amino acid.
  • the process is repeated to produce a second library of translated proteins comprising a second label, e.g., by using a different modified tRNA species having a different modified tRNA.
  • biotin-lys-tRNA is used for one translation and BODIPY®FL-lys-tRNA for the other.
  • Proteins from the first library are mixed with proteins from the second library and the formation of binding complexes is measured to identify pairs of proteins that have binding affinity for each other.
  • the translation reactions and/or binding assays are preferably carried out in parallel in a multi-well plate format.
  • protein:protein interactions are detected in the following way.
  • Two translation products from the two different libraries are mixed together to form a mixture of the two differently labeled translation products. These are allowed to bind and the resulting mixture is then captured on to a solid phase using a binding species specific for one of the labels in the products of the translation (alternatively, one of the translation products is first captured and is then contacted with the other translation product).
  • a labeled or detectable binding species is added which recognizes the label not used to immobilize the product of the translation reaction (alternatively, this label is a directly detectable label and is detected directly). In this way the interaction between the proteins produced in the two translation reactions is detected.
  • Another example of this is where a gene is transcribed and translated in the presence of Ru(bpy) 3 2+ -lys-tRNA and a gene is transcribed and translated in the presence of biotin-lys-tRNA. These reactions result in the production of proteins that are labeled with Ru(bpy) 3 2+ and biotin respectively. These two labeled proteins in the translation mix are mixed and allowed to interact. These proteins are then captured on to a solid phase using for example streptavidin followed by detection of the Ru(bpy) 3 2+ to allow the determination of protein:protein binding.
  • clones are preferably either obtained or produced in a desired cloning vector or nucleic acid construct.
  • the DNA from these is then subjected to an in vitro transcription reaction and followed by an in vitro translation reaction. These two reactions can also be combined in a single in vitro reaction.
  • the protein produced is also labeled using a modified tRNA that directs the incorporation of a modified amino acid during this step.
  • the reaction mix or alternatively a reaction mix obtained by other means, is then subjected to assays for nucleic acid sequence binding activity.
  • the protein in the mix is captured on to a solid phase using a binding protein specific for the modification introduced by the modified tRNA during the translation reaction.
  • biotin-lys-tRNA that results in a biotinylated protein containing lysine residues modified with biotin.
  • the biotinylated proteins produced in this way are then captured on to a solid phase using an avidin coated solid phase.
  • nucleic acid sequence binding activities are determined using various methods including using a labeled nucleic acid sequence.
  • nucleic acid sequence is added to the proteins captured on to the solid phase and incubated to allow binding. Following this step the degree of binding is determined based on a determination of the amount of label captured on to the solid phase.
  • the nucleic acid sequence is a DNA sequence that has been labeled with an ECL label during synthesis using ORI-TAG® labelled phosphoramidite (IGEN International, Inc., Gaithersburg, MD).
  • the labeled nucleic acid is added to the translation reaction prior to immobilization of the translation product on to a solid phase.
  • clones are either obtained or produced in a desired cloning vector or nucleic acid construct.
  • the DNA from these is then subjected to an in vitro transcription reaction and followed by an in vitro translation reaction. These two reactions can also be combined in a single in vitro reaction.
  • the protein produced is also labeled using a modified tRNA that directs the incorporation of a modified amino acid during this step.
  • the reaction mix is then subjected to assays for nucleic acid sequence binding activity.
  • the binding activity of the protein from the transcription translation system is analyzed by determining its binding to an immobilized nucleic acid sequence.
  • a potential nucleic acid sequence is immobilized on to a solid phase using methods known in the art such as passive adsorption, covalent linkage to a chemically activate surface including NHS ester, epoxide, cross-linking reagents (see above), maleimide chemistries.
  • the nucleic acid sequence are first coupled using typical chemistries such as NHS ester, epoxide, maleimide to a carrier typically a large molecule that can then be immobilized using a simple passive adsorption methods. This method also allows for the construction of arrays of multiple nucleic acid sequences within a single container.
  • the labeling of the nucleic acid either during synthesis or post synthesis with a small molecule binding species such as biotin, fluorescein, digoxigenin also allows for the immobilization of the nucleic acid on to a solid phase in specific arrays using an immobilized binding partner.
  • a small molecule binding species such as biotin, fluorescein, digoxigenin
  • the labeled proteins that have been produced in the transcription and translation reaction are contacted with this coated solid phase.
  • the binding activity of the proteins from the transcription and translation reactions is determined based on the detection of the modified amino acid incorporated during the translation reaction. Typically this is achieved using labeled; avidin, streptavidin or an antibody specific for the modification on the modified amino acid.
  • An alternative embodiment of the invention is directed to the discovery of antigens recognized by antibodies.
  • These antibodies may be monoclonal or polyclonal or patient samples, i.e., serum or plasma. These patients may be animal or human and may or may not have been diagnosed with a disease or medical condition. Examples of patient who have been diagnosed with a disease or medical condition include auto-immune disease, immunological disorders, asthma, heart disease, MS, atherosclerosis and cancer. Screens for antigens have been made typically using bacterial expression methods; this invention includes novel methods for screening for antigens using proteins (preferably, labeled) produced in a transcription and translation reaction. The proteins are contacted with the antibodies and the formation of binding complexes is measured.
  • proteins preferably, labeled
  • the binding measurements are carried out in a solid phase format and involve the capture of the translated proteins and detection of the antibody or, alternatively, capture of the antibody and detection of the translated proteins.
  • the antibodies and/or proteins are, preferably, labeled and captured and/or detected via these labels.
  • the antibodies are captured or detected using antibody-specific binding reagents such Proteins A, G or L or secondary antibodies.
  • translated proteins are immobilized on to a solid phase followed by detection of those binding to the added antibodies using either directly labeled antibodies or via a labeled binding species specific for the added antibody.
  • the proteins produced in the transcription and translation reaction are immobilized on to a solid phase in a multiwell plate as described above.
  • the proteins are contacted with the antibodies and the formation of binding complexes is detected.
  • Each protein may be analyzed in individual wells of a multi-well plate.
  • an array of proteins is produced in a well of a multi-well plate or on a protein chip and a plurality of binding assays are carried out in one reaction volume.
  • the proteins produced in the translation are labeled using a modified precharged tRNA such as biotin-lys-tRNA as described earlier.
  • These labeled proteins are then immobilized by binding to avidin or streptavidin immobilized on to a carbon containing surface or magnetic beads. These immobilized proteins are thus available for binding to the antibodies to allow the detection and determination of the genes and proteins of the antigens recognized by the antibodies.
  • the antibodies bound to the immobilized antigen are detected using for example ECL labeled anti antibodies (IGEN International, Inc., Gaithersburg, MD).
  • certain embodiments of the invention preferably involve expression of a protein using a cell-free translation system, immobilization of the protein (preferably, via a label introduced into the protein during the translation reaction) and assaying the immobilized protein for an activity (e.g., a catalytic activity, a substrate activity, a binding activity, etc.).
  • an activity e.g., a catalytic activity, a substrate activity, a binding activity, etc.
  • a biotin-labeled protein was produced by transcription/translation in a rabbit reticulocyte system that was supplemented with tRNA that was charged with biotin-lys, the protein was immobilized on a streptavidin-coated surface and the immobilized protein was assayed for kinase activity by measuring its ability to phosphorylate poly(Glu,Tyr).
  • the mechanism of this effect has been shown to be the recruitment of endogenous kinases in rabbit reticulocyte lysate through binding to the biotin-labeled protein; the observed activity can be significantly reduced or eliminated through the use of high stringency washes designed to dissociate the binding interaction responsible for the recruitment.
  • a translated protein or alternatively a protein obtained by other means, is immobilized, the protein is not washed or washed with a low stringency wash, and the protein is assayed for an activity of interest or for its ability to recruit a species having an activity of interest.
  • a translated protein is immobilized, the protein is washed with a high stringency wash, and the protein is assayed for an activity of interest in the absence of recruited species.
  • a translated protein is i) immobilized, ii) the protein is not washed or washed with a low stringency wash, iii) the protein is assayed for an activity of interest or its ability to recruit a species having an activity of interest (e.g., by introduction of a substrate for an enzymatic activity), iv) the protein is washed in a high stringency wash and v) the protein is assayed for the activity of interest in the absence of recruited species.
  • a translated protein is i) immobilized, ii) the protein is not washed or washed with a low stringency wash, iii) the protein is assayed for an activity of interest or its ability to recruit a species having an activity of interest (e.g., by introduction of a substrate for an enzymatic activity), iv) the protein is washed in a high stringency wash and v) the protein is assayed for the activity of interest in the absence of recruited species.
  • the high stringency wash is a high salt buffer, preferably, having an salt concentration of greater than or equal 750 mM, or more preferably 1 M.
  • high stringency may be accomplished through non-physiological pH (preferably > 8.5 or ⁇ 6) or the introduction of denaturing detergents or chaotropic agents.
  • the label used in the translation reaction is preferably chosen for its ability to participate in binding reaction that is relatively insensitive to high stringency conditions (e.g., biotin-avidin or biotin-streptavidin).
  • the identity of recruited species may be identified through a variety of analytical techniques including sequencing, mass spectrometry, capillary electrophoresis, HPLC, electrophoresis, western blotting, antibody arrays, or a combination thereof.
  • the in vitro transcription and translation reaction can be carried out in the presence of a membrane preparation (e.g., microsomes) that allows for the signal peptide processing, insertion into membranes and glycosylation of the proteins produced by the in vitro translation reaction.
  • a membrane preparation e.g., microsomes
  • membrane preparations include canine microsomal membranes ( Walter, P. and Blobel, G. (1983) Meth. Enzymol. 96, 84 ), Xenopus egg extracts ( Zhou, X, et al (200) In Vitro Cell. Dev.Biol. -Animal 36, 293-298 ; US 6,103,489 ).
  • ligands may be drugs, proteins, lipids, phospholipids etc., where the receptor is thought to be a membrane bound protein.
  • the proteins may be immobilized and/or analyzed according to the methods of the invention while membrane bound. Alternatively, they are released from the membranes prior to processing or analysis.
  • the microsomal membranes are, preferably, lysed using detergents such as 1% Triton X-100. This step allows the binding, enzyme, and/or substrate activity of the proteins to be fully accessed.
  • This lysis step may be carried out either before or after immobilization (using, e.g., the methods based on the modified amino acids precharged on tRNA, or affinity tags included in the expressed protein, or directly immobilized by passive adsorption.
  • the proteins produced in the transcription translation reaction are produced with epitope tags or affinity tags that are incorporated into the coding sequence of the various genes of interest. This can be used in place of the modified tRNA to label the products of the translation reaction.
  • epitope tags can thus be used to bind to solid phases and for detection via the use of specific binding partners to these epitope tags.
  • the specific binding partners are typically antibodies but may include other classes of binding partners. Examples of useful affinity tag/binding partner pairs include GST:glutathione, Peptide:avidin, MBP:maltose, DNA binding protein:DNA, His 6 :Ni-NTA.
  • proteins produced in the transcription and translation reaction are immobilized directly to the solid phase via various methods such as passive adsorption, or coupling to chemically activated surfaces using various chemistries such as NHS ester, epoxide and maleimide. Proteins immobilized in this way are thus available for detection of specific interactions or reactions.
  • the translation step is omitted from methods of the invention that employ transcription and translation.
  • mRNA coding for an amino acid sequence of interest preferably purified mRNA
  • a translation system preferably a cell-free translation system
  • the assays of the invention are conducted on proteins that are not generated by in vitro translation, preferably labeled proteins. These may be proteins that are purified from natural sources or expressed in living cells.
  • these various materials and methods are all handled in multiwell plates.
  • these plates typically have 20 or more wells, 40 or more wells, 84 or more wells, 96 or more wells, 3 84 or more wells or 1536 or more wells.
  • an assay technique is used to measure a known activity of a protein. It is understood that the same technique will be applicable for identifying other proteins that have these or similar activities, in particular, proteins in which these activities have not been previously identified.
  • Compound 1 pictured below is the NHS ester of an electrochemiluminescent label used to label biomolecules for electrochemiluminescence measurements. Labeling of biomolecules was carried out by adding Sulfo-TAG NHS Ester to a solution of the biomolecule in phosphate buffered saline, pH 8.0. The labeled biomolecules were typically purified from unbound label by size exclusion chromatography (using, e.g., Superdex Peptide Gel or Sephadex G50, Pharmacia Biosciences) or reverse phase HPLC.
  • the ratio of labels per protein was calculated from the concentration of labels (calculated from the extinction coefficient of Sulfo-TAG label at 455 nm, ⁇ 455 ⁇ 13,700 M -1 cm -1 ) and the concentration of protein (determined using the BCA Protein Assay, Pierce Chemicals).
  • Electrochemiluminescence measurements were carried out using screen-printed carbon ink electrodes patterned on the bottom of specially designed 96-well multi-well or multi-domain multi-well plates.
  • the multi-well plates are described in more detail in copending Application No. 60/301,932 (entitled “Assay Plates, Reader Systems and Methods for Luminescence Test Measurements", filed on June 29, 2001) and particularly in the description of Plate Type B in Example 6.1 (IPR plate) and U.S. Application Serial Nos. 10/185,274 and 10/185,363, filed June 28, 2002 , each hereby incorporated by reference.
  • Each well of the plate has a patterned working electrode comprising 1, 4, 7, or 10 assay domains (roughly in the center of the well) that are exposed regions of electrode surface that are defined by a patterned dielectric layer on the electrode surface.
  • the dielectric layer could be used to confine small volumes of liquid to specific assay domains (e.g., to allow for confinement of an immobilization reagent to the working electrode surface).
  • Each well also has two counter electrodes surfaces (roughly at two edges of the well).
  • the carbon ink electrodes were treated with an oxygen plasma to increase the surface area of exposed carbon particles and to improve the wettability of the surface (plasma treatment was not required for carrying out the assays, however, in some applications plasma treatment was found to improve the ratio of signal to background as well as assay precision).
  • the plates were coated with proteins, such as streptavidin, avidin, or an antibody by depositing an aliquot of a protein stock solution (preferably 0.1-0.5 mg/ml in PBS, pH7.4, 0.01% Triton-X100) onto the working electrode surface of each well of 96-well IPR plate and air-drying for 5 hours. Following the coating, the plate was blocked overnight with a blocking solution (containing BSA in a PBS buffer with stabilizers) at 4°C followed by three washes with PBS.
  • proteins such as streptavidin, avidin, or an antibody by depositing an aliquot of a protein stock solution (preferably 0.1-0.5 mg/ml in PBS, pH7.4, 0.01% Triton-X100) onto the working electrode surface of each well of 96-well IPR plate and air-drying for 5 hours. Following the coating, the plate was blocked overnight with a blocking solution (containing BSA in a PBS buffer with stabilizers) at 4°C followed by three washes with PBS.
  • the plates were washed 3 times with 300 ⁇ l of a solution containing 2 % Sucrose, 0.1% Tween, 0.2% Kathon, 2.3 % Ammonium Dihydrogen Phosphate, dried in a vacuum chamber for 3.5 minutes, placed in foil pouches containing desiccant, vacuum sealed and stored at 4C. Just prior to use, these plates were blocked with 300 ⁇ l of the BSA-containing blocking solution overnight at 37°C followed by three washes with water.
  • electrochemiluminescence from ECL labels on the surface of the carbon electrodes was induced and measured using an imaging plate reader as described in Examples 6.1 and 6.3 of copending Provisional Application No. 60/301,932 (entitled “Assay Plates, Reader Systems and Methods for Luminescence Test Measurements", filed on June 29, 2001, hereby incorporated by reference) and U.S. Application Serial Nos. 10/185,274 and 10/185,363, filed June 28, 2002 , each hereby incorporated by reference. Analogous plate readers are now commercially available (Sector HTSTM instrument, Meso Scale Discovery).
  • Binding Buffer 1 refers to PBS, pH7.4 containing 0.1%BSA, 0.1% bovine IgG, 0.2% tween-20, and protease inhibitors (protease inhibitor cocktail, EDTA free, Roche Applied Science).
  • Binding Buffer 2 refers to 25 mM Tris-HCl buffer pH 7.4 containing 100 mM NaCl, 0.05 mM Na 3 VO 4 , 0.004% TritonX-100, 2 mM DTT and protease inhibitors (protease inhibitor cocktail, EDTA free, Roche Applied Science). Proteins were produced by coupled transcription/translation using TnT reaction mixes (Promega Corp, WI).
  • the TNT Quick coupled transcription-translation system contains rabbit reticulocyte lysate pre-mixed with most of the reaction components necessary to carry out transcription and translation in vitro (TNT mix), including all the amino acids except methionine.
  • biotin-labeled in vitro translated proteins were produced in reaction mixtures that contained 8-30 ⁇ g/ml of a selected plasmid encoding protein of interest, 5 ⁇ l of TNT T7 Quick reticulocyte lysate mix (Promega Corp, WI) or TnT SP6 Quick Coupled Transcription / Translation System (Promega Corp, WI), 20 ⁇ M methionine, 50 ⁇ M MG-132 (Calbiochem, proteasome inhibitor to inhibit degradation of ubiquitylated proteins by proteasome) and 20 ⁇ g/ml biotinylated Lys-tRNA (transcend tRNA, (Promega).
  • BB1 was used in screens involving the ubiquitination pathway.
  • BB2 was used for other assay formats. The produced proteins were used in the assays of interest with or without further purification.
  • the example demonstrates the detection of ubiquitylated proteins produced by in vitro translation by adding labeled ubiquitin in the translation reaction.
  • RGS4 was produced by transcription/translation of 30 ⁇ g/ml plasmid pcDNA3-RGS4 ( Davydov and Varshavsky, 2000, J. Biol. Chem., 275:22931-22941 ) under the conditions described in Materials and Methods except for the addition of 1 ⁇ M Ub aldehyde (Calbiochem, to inhibit deubiquitylating enzymes) and 5 ⁇ M Sulfo-Tag-labeled Ub (having an average of 4.1 labels per Ub molecule). Then 3 ⁇ l of the reaction was mixed with 50 ⁇ l of BB1 in a 96-well streptavidin-coated non-plasma treated IPR (NPT IPR) plate.
  • NPT IPR non-plasma treated IPR
  • the plate was left on a tabletop shaker for 1 hour to allow for binding of biotinylated proteins to the surface. Thereafter the plate was washed three times with PBS followed by the addition of 100 ⁇ l of ORIGEN® Assay Buffer (IGEN International, Inc., Gaithersburg, MD) into each well, and ECL signals were measured using an imaging plate reader (IPR).
  • IPR imaging plate reader
  • the example demonstrates detection of ubiquitylated proteins produced by in vitro translation, using immobilized antibodies able to bind the ubiquitin post-translational modification.
  • Transcription-translation reaction mixtures were prepared as described in the Example 1 except that no labeled ubiquitin was added. Some reactions also contained 1mM dipeptide inhibitor of the N-end rule pathway (either Arg- ⁇ -Ala, or Trp-Ala) together with 50 ⁇ M bestatin as indicated (Table 2). The reaction was allowed to proceed for 20 min at 30°C.
  • the example demonstrates detection of ubiquitylated proteins produced by in vitro translation, using labeled antibodies specific to the Ub post-translational modification.
  • the protein was prepared as described Example 2 and mixed with 60 ⁇ g/ml Sulfo-TAG-labeled FK2 antibody (Affiniti) having 5.6 labels per antibody, in a streptavidin-coated NPT IPR plate.
  • the plate was left on a tabletop shaker for 1 hour to allow for biotinylated proteins to bind to the surface of the plate and for the antibody to bind to ubiquitylated proteins. Thereafter the plate was washed three times with PBS followed by the addition of 100 ⁇ l of ORIGEN assay buffer (IGEN International, Inc., Gaithersburg MD) into each well, and ECL signals were measured using an imaging plate (See, Example 1).
  • EXAMPLE 4 Detection Of Ubiquitinated Proteins Produced In A Transcription And Translation Reaction By Capture With An Anti-Ubiquitin Antibody Immobilized On To A Multiwell Plate.
  • EST clones in bacteria were obtained: IMAGE ID #, 3446518, 3446518, 3446518, 3914731 (RGS4), 3914731 (RGS4), 3914731 (RGS4) (Incyte, Palo Alto).
  • Bacteria containing EST clones were grown in 2 ml LB with 50 ⁇ g/ml Amp, and DNA was isolated using QIAprep Spin Miniprep Kit (Qiagen). DNA yield was 5-20 ⁇ g.
  • Proteins were prepared from the EST clones in transcription-translation reaction mixtures (total volume 12.5 uL) as described in the Materials and Methods except for the addition of 1 ⁇ M Ub aldehyde (Calbiochem). Then 1 ⁇ l of the reaction product was mixed with 50 ⁇ l of BB1 supplemented with 20 mM AMP-PNP, 20 mM N-ethylmaleimide and 2 ⁇ g/ml Sulfo-TAG-labeled streptavidin in to each well of an IPR plate coated with FK2 antibody.
  • the plate was left on a tabletop shaker for 1 hour to allow for ubiquitylated proteins to bind to the surface of the plate and for the Sulfo-TAG-labeled streptavidin to bind to biotinylated proteins. Thereafter the plate was washed three times with PBS followed by addition of 100 ⁇ l of ORIGEN Assay Buffer (IGEN, International, Inc., Gaithersburg MD) into each well, ECL signals were measured using an imaging plate reader.
  • ORIGEN Assay Buffer IGEN, International, Inc., Gaithersburg MD
  • RGS4 is a substrate of the N-end rule-dependent ubiquitylation in reticulocyte lysates ( Davydov and Varshavsky, 2000, J. Biol. Chem., 275:22931-22941 ).
  • the ECL ubiquitylation assay was performed in the presence of dipeptide inhibitors Arg- ⁇ -Ala (type 1 inhibitor of the N-end rule pathway), or Trp-Ala (type 2 inhibitor of the same pathway).
  • the RGS4 signal was reduced 3-4 fold in the presence of Arg- ⁇ -Ala (a type 1 inhibitor of the N-end rule pathway), but not in the presence of Trp-Ala (a type 2 inhibitor of the N-end rule pathway) in agreement with the previous report that RGS4 is a type 1 N-end rule substrate in reticulocyte lysates ( Davydov and Varshavsky, 2000, J. Biol. Chem., 275:22931-22941 ). In contrast, the dipeptides had little effect on the assay signal from clone 3446518 ( Fig. 2 ). Thus, a different pathway of the ubiquitin system mediates ubiquitylation of the protein produced from clone 3446518.
  • Transcription-translation reaction mixtures contained components described in Materials and Methods and 400 ⁇ Ci/ml 35 S-methionine (AmershamPharmaciaBiotech), 1 ⁇ M Ub aldehyde (Calbiochem) in a total volume of 12.5 ⁇ l. The reaction was allowed to proceed for 45 min and was stopped by addition of 4-fold excess of SDS-gel loading buffer (Invitrogen) with 5% ⁇ -mercaptoethanol.
  • the Example describes detection of ubiquitinated proteins produced in a transcription and translation reaction via capture onto magnetic beads in a multiwell plate format.
  • the example shows detection of ubiquitinated proteins produced in a transcription and translation reaction via capture on to magnetic beads in a multiwell plate format.
  • ubiquitination is detected using labeled antibody.
  • Transcription-translation reaction mixtures containing pcDNA3-RGS4 or pcDNA3-RGS4-C2V plazmids( Davydov and Varshavsky, 2000, J. Biol. Chem., 275:22931-22941 ) were prepared and used as described in the Example 2.
  • the reaction products were mixed with 3 ⁇ g Sulfo-TAG labeled FK2 antibody in a 96-well plate in a binding buffer.
  • Sulfo-TAG labeled FK2 antibody had 5.6 Sulfo-TAG labels per antibody molecule.
  • the plate was left on a tabletop shaker for 1 hour to allow for binding of the antibody to ubiquitylated proteins.
  • EXAMPLE 7 Assay To Screen For Tyrosine Kinase Activity.
  • the example demonstrates an ORIGEN based HTS assay for tyrosine kinase activity of fer (fms/fps related) protein tyrosine kinase, testis specific 2 (Fert2) expressed in reticulocyte lysate.
  • the assay format developed for this purpose utilizes following steps:
  • the example demonstrates an ORIGEN based HTS assay for autophosphorylation activity of Spleen Tyrosine kinase and Eph receptor-A7 expressed in reticulocyte lysate.
  • SYK Spleen Tyrosine Kinase
  • EphA7 Eph receptor-A7
  • ECL signal produced by autophosphorylation activity of tyrosine kinases expressed in 0.5 ⁇ l of TnT reaction was about 25 fold (in SYK) or 47 fold (in EphA7) higher than the background noise produced by Mock (Luciferase) (see Table 11).
  • the example demonstrates an ORIGEN based HTS assay for SRC phosphorylation of SKAP-HOM in reticulocyte lysate using Sulfo-TAG labeled anti-phosphotyrosine.
  • Tyrosine kinases may phosphorylate protein substrates with high specificity. However, the map of such substrates for each of the known kinase is far from being complete.
  • the assay format developed for this purpose utilizes following steps:
  • SRC specifically phosphorylates SKAP-HOM, a protein that was predicted to be a potential substrate based on its homology to known SRC substrates ( Curtis DJ, et al (2000) Exp Hematol, 28, 1250-9 ; Marie-Cardine A, et al (1998) FEBS Lett, 435, 55-60 ).
  • the control proteins SYK or Luciferase (Mock) did not show significant levels of phosphorylation by SRC.
  • the non-specific tyrosine phosphorylation of Mock, SYK or endogenous substrates of the Tn T system were of considerable lower magnitude, about 17 fold lower to that of the SKAP-HOM (Table 12).
  • Example of ECL based assays for phosphopeptide and protein interaction The model assay measures the interaction between ⁇ TRCP and phosphorylated IkBa peptide captured on a solid phase.
  • a phosphopeptide derived from the IkBa degradation motif [SEQ ID No. 1] and a phosphopeptide derived from c-Myc [SEQ ID No. 4] were chemically synthesized (New England Peptide, Fitchburg MA) with the following sequences respectively:
  • the cystine residues at the C- or N-terminuses were added to the sequences to allow for covalent coupling of the peptide to a carrier protein.
  • the IkBa peptide [SEQ ID No.1] contains the six-residue motif with "DSGXXS" consensus sequence that confers the binding to the ⁇ TRCP, an F-box/WD40 repeat containing protein.
  • the binding of the peptide to ⁇ TRCP is absolutely dependent on the presence of the two phosphoserine residues. Singly phosphorylated peptide or phosphothreonine substitution abolish interaction.
  • the c-Myc derived phosphopeptide [SEQ ID No. 4] was used as a control.
  • a maleimide pre-activated BSA (Pierce, Pittsburgh PA), which contains estimated 17 active maleimide groups on the protein surface, was used as the protein carrier, and was supplied as a lyophilized preparation.
  • Coupling of the IkBa peptide [SEQ ID No.1] to the pre-activated BSA was carried out with 2 mg BSA (30 nmol) and 17 equivalents of the 25 residue phosphopeptide in 400 ⁇ l PBS.
  • the reaction was terminated with 2 mM L-cystine after incubation at room temperature for 30 minutes.
  • the peptide:BSA conjugate was then purified on PD-10 gel filtration column to remove uncoupled peptide and analyzed with SDS PAGE to monitor the coupling reaction.
  • ⁇ TRCP1 and ⁇ TRCP2 Two ⁇ TRCP genes, ⁇ TRCP1 and ⁇ TRCP2, are present in the human genome on chromosome 5 and 10, respectively.
  • the coding sequence of these two genes shares over 90% identity and several full-length cDNAs of which are available as EST clones from Incyte (Palo Alto, CA) IMAGE #3491843 and 4237375. Also included were three other non specific EST clones as controls (Incyte, Palo alto, CA). Plasmid DNAs from these EST clones and other clones were prepared using the Qiagen miniprep kit (Qiagen, Los Angeles, CA). These DNAs were used to produce the ⁇ TRCP protein and proteins encoded by the other DNAs as described in the Example 7.
  • Proteins were transferred into wells of the peptide-coated and BSA-blocked IPR plates. Sulfo-TAG labeled streptavidin (50 to 100 ng/well) was added to each well as the detection agent. The binding reaction can be carried out in cold room for over two hours or at room temperature for 1 hour without significant difference. After binding, the IPR plates were washed twice with PBS and 100 ⁇ l of ORIGEN assay buffer (IGEN International, Inc., Gaithersburg, MD) was added to each well before reading the ECL signal. The ECL signals were measured using an imaging plate reader (IPR).
  • IPR imaging plate reader
  • EXAMPLE 11 Assay For Interaction Among Multiple Proteins And Their Cognate Precognition Phosphopeptide Arrayed On Multiple Spots In A Single Well.
  • peptide-BSA conjugates were then deposited on the assay domains of a 4-spot multi-array IPR plate (i.e., in one of the four exposed regions of working electrode defined by the dielectric layer) with an automated micro-dispensing system (Bio-Dot Dispenser, Bio-Dot, Irvine CA). Typically about 120 fmole of peptide:BSA conjugate in 0.25 ⁇ l was deposited on each spot of a 4-spot multi-array IPR plate. These plates are dried then used or stored under desiccated conditions. A few hours prior to usage the plates were rehydrated and blocked with 3% BSA in PBS.
  • ECL signals imaged from spot 1(IkBa-24pp-BSA conjugate) were 100-fold higher in wells containing ⁇ TRCP1 and ⁇ TRCP2 than in wells derived from TnT reaction mixtures containing no exogenous DNA.
  • spots 2-4 in the same wells gave low ECL signals.
  • each peptide not only serves as the capture agent for assaying specific interaction with its binding protein, but also at the same time serves as a control for other peptide-protein interaction to be determined in the same well.
  • the same principle was illustrated once again by the binding ofGrb2 and PLC ⁇ proteins with EGFR carboxyl terminus phosphotyrosine peptides.
  • This example demonstrates a mulfi-plexed approach for screening for proteins that bind phosphopeptides.
  • the assay used an array of multiple phosphopeptides for identifying multiple protein-peptide interactions in a single well and used a coded multiplex approach.
  • peptides were chemically synthesized (New England Peptide, Fitchburg MA). The peptides were coupled to pre-activated maleimide BSA according to the procedure described in Example 11.
  • EGFR-pTyr992 [SEQ ID No. 5] DADE(p)YLIPQQGFFSSPSTSC EGFR-pTyr1068 [SEQ ID No. 7] LPVPE(p)YINQSVPKRPAGSVC EGFR-pTyr1148 [SEQ ID No.
  • KGSHQISLDNPD(p)YQQDFFPKEAKPNC IkBa-24ppC [SEQ ID No.1] LKKERLLDDRHD(p)SGLD(p)SMKDEEYC Myc-p58Tp62S [SEQ ID No.4] CPSEDIWKKFELLP(p)TPPL(p)SPSRRSGL Smad3C [SEQ ID No. 11] CGPLQWLDKVLTQMGSPHNPIS(p)SV(p)S
  • the peptides EGFR-pTyr992 [SEQ ID No. 5], EGFR-pTyr1068 [SEQ ID No.7] and EGFR-pTyr1148 [SEQ ID No.9] were derived from the phosphorylation sites in EGF receptor.
  • Peptide IkBa-24ppC [SEQ ID No. 1] is described in Example 10.
  • the peptides Myc-p58Tp62S [SEQ ID No. 4] and Smad3C [SEQ ID No. 11] are derived from c-Myc and SMAD3.
  • certain binding partners are known (Table 16).
  • Plasmid DNAs from these various clones were prepared using the Qiagen miniprep kit (Qiagen, Los Angeles, CA). These DNAs were used to produce proteins as described in the Materials and Methods. Proteins were transferred to a well of the 4 spot peptide-coated and BSA blocked ECL plates. The binding assay (using Sulfo-TAG labeled streptavidin as a detection agent) was carried our according to the procedures described in Example 11.
  • ECL signals for each pooled peptide spot are presented in Table 18.
  • Table 18 also lists the code (i.e., pattern of positive spots) obtained with each protein.
  • the identity of the binding peptide as determined by the code is presented in Table 19.
  • Table 18. Spot 3491843 4237375 4398016 4419252 3872466 3907115 Blank ⁇ TRCP1 ⁇ TRCP2 Grb2 PLC ⁇ CBP Shc1 A 22 9536 7791 36 239 19 260 B 54 208 105 38 2783 30 318 c 28 111-84 8615 509 1550 22 24 D 20 171 74 386 3388 15 26 Code Neg. A,C A,C C, D B,C,D Neg. A, B Table 19.
  • EST clones IMAGE #'s 3886018, 3910505 and 3915089 were obtained from Incyte (Palo Alto, CA). Plasmid DNA was prepared from these EST clones using QIAprep96 Turbo Miniprep (cat#27191, QIAgen, Los Angeles, CA), according to manufacturer's instructions. cDNA was eluted using 150 ⁇ l buffer EB (QIAgen).
  • the proteins were produced in the transcription translation (TnT) reaction as described in the Materials and Methods.
  • EXAMPLE 14 Assay To Detect Binding Species Produced By In Vitro Translation Using A Magnetic Bead Based Assay System (Prophetic).
  • peptides are immobilized on to Dynal M280 beads coated with streptavidin making use of the well known streptavidin biotin interaction.
  • the peptide is dissolved in PBS, 0.1% NP40 and incubated with the beads for 1hour followed by 4 washes into binding buffer (50mM Tris-HCL pH7.5, 300mM NaCl, 2mM EDTA, 0.1% NP40 and protease inhibitor cocktail (Roche)).
  • the TnT reaction are carried out following the manufactures protocol.
  • the reaction is was as follows, 25 ⁇ l of lysate (coupled TnT, Promega, Madison WI), 0.5 ⁇ g of plasmid DNA.
  • the plasmid DNA contains the ⁇ TRCP gene fused in frame with the Flag epitope and contains a RNA polymerase site.
  • a plasmid vector directing transcription of flag- ⁇ TRCP gene fusion is constructed using synthetic PCR primers to the EST clone IMAGE # 3491843. The primers TATGTCGACATGGATTATAAGGATGACGATGACAAAGACCCGGCAGAGGCG GTGCTG [SEQ ID No.
  • TATGCGGCCGCTTATCTGGAGATGTAGGTGTA (SEQ ID No. 14] are used to generate the flag- ⁇ TRCP gene fragment of about 1750 bases.
  • This PCR DNA product is then subject to restriction digestion with Sal1 and Notl following the manufactures protocol (New England Biolabs, Beverly, MA).
  • the cut PCR DNA product is gel purified and ligated into Sal1 and Not1 cut pCMV-SPORT6 (Invitrogen, Carlsbad, CA). The ligation is transformed into E.coli and the clones screened for the correct insert.
  • One of the resulting clones is used in the TnT reaction to produce the fusion protein Flag- ⁇ TRCP. Protein expression in the TnT reaction is confirmed using radioactive Met following the manufactures protocol (Promega, Madison, WI). To determine if we can detect the binding of flag- ⁇ TRCP to the phosphopeptide we incubate the products of the TnT reaction specific for the flag- ⁇ TRCP with beads coated with the consensus phosphopeptide or the control peptide. These beads are washed and ORI-TAG® (IGEN International, Inc., Gaithersburg, MD) labeled anti Flag antibody is added in binding bluffer and then incubated followed by analysis in an ORIGEN M8 (IGEN International, Inc., Gaithersburg, MD).
  • ORI-TAG® IGEN International, Inc., Gaithersburg, MD
  • EXAMPLE 15 Assay For DNA Binding Proteins Produced In A Tnt Reaction (Prophetic).
  • cDNA clones are subjected to a TnT reaction followed by the detection of those proteins that are able to bind specifically to a given nucleic acid sequence.
  • the following EST clones coding for the YY1 transcription factor are tested: IMAGE IDs, 3987868, 3156776, 2655378, and 3859359 (Incyte Genomics).
  • IMAGE IDs 3987868, 3156776, 2655378, and 3859359
  • the luciferase plasmid included in the TnT kit from Promega is used. DNA is prepared from these clones using the Qiagen miniprep kit (Qiagen, Los Angeles, CA).
  • the oligo specific for YY1 is made synthetically (IGEN International, Inc., Gaithersburg, MD) using the following sequences 5'ORI-tag-labeled- ACGTACGTAC CGCTCCGCGGCCATCTTGGCGGCTGGT [SEQ ID No. 15] and its complement 5'ACCAOCCGCCAAGATGGCCGCGGAGCGGTACGTACGT [SEQ ID No. 16]. These two oligonucleotides are annealed by mixing together equimolar amounts in 10mM Tris-HCL pH 7.9, 1mM EDTA, 50mM KCl. This double stranded oligonucleotide (ECL-oligo) is then used in the following DNA binding assay.
  • ECL-oligo double stranded oligonucleotide
  • the YY1 protein and the control luciferase are produced in a coupled TnT reaction, as described in the Materials and Methods. Then 5 ⁇ l of the reaction product is mixed with, 50 ⁇ l of binding buffer and 28-14ng/ml 5'ORI-tag-labeled double stranded oligonucleotide produced above, in a streptavidin-coated NPT IPR plate (see examples above). The plate is left on a tabletop shaker for 1 hour to allow for biotinylated proteins to bind to the surface of the plate and for the ECL-oligo to bind to the biotinylated proteins.
  • ECL signals are measured.
  • the ECL signals are measured using an imaging plate reader (IPR).
  • IPR imaging plate reader
  • EXAMPLE 16 Assay For DNA Binding Proteins, Using A Multi-SpotTM Approach (Prophetic).
  • the DNA sequences listed in Table 21 are synthesized as well as the complementary sequences without 5' modifications. These oligos are rendered double stranded using the method described above by mixing the complementary pairs together.
  • the SH-X- group at the 5' end of the oligonucleotide sequence is introduced during synthesis using a cycle of Spacer Phosphoramidite 18 (Catalog Number: 10- 1918 -xx Glenn Research, Herndon, VA) followed by a cycle of Thiol-Modifier C6 S-S (Catalog Number: 10-1936-xx Glenn Research, Herndon, VA) during synthesis. This results in a 5' free SH group attached to a hexaethyleneglycol spacer ready for coupling.
  • the SH-X- moiety at the 5' terminus is added to the sequence to allow for covalent coupling of the DNA to a carrier protein.
  • a maleimide pre-activated BSA (Pierce, Pittsburgh PA) is used as the protein carrier.
  • the protein contains about 17 active maleimide groups on the protein surface and is supplied as lyophilized preparation.
  • the coupling reaction is carried out with 2 mg BSA (30 nmol) and different molar amount of the DNA in 400 ⁇ l PBS.
  • the molar ratio of DNA relative to the BSA is between 4:1 to 17:1.
  • the reaction is terminated with 2 mM L-cystine after incubation at room temperature for 30 minutes.
  • the DNA:BSA conjugate is then purified by gel filtration to remove uncoupled DNA and analyzed with SDS PAGE to monitor the coupling reaction. After coupling and purification, the column fractions containing the DNA:BSA conjugates are pooled and the protein concentration of the pooled samples is determined by BCA assay (Pierce, Pittsburgh PA). NaN3, (0.02%) is added to the final peptide;BSA conjugate sample for long term storage at 4 °C.
  • DNA for the EST clones encoding c-jun, glucocorticoid receptor, p53 and CDP are isolated using QIA-prep column (QIAGEN, Los Angeles, CA). These DNA molecules are used to produce the proteins in a Quick coupled SP6 in vitro transcription and translation (TnT) reaction (Promega, Milwaukee, WI) as described in Materials and Methods. The proteins are transferred to a well of the 4 spot DNA:BSA coated and BSA blocked IPR plates. To these wells 50 to 100 ng/well of Sulfo-TAG labeled Streptavidin (see example 2) is added as the detection agent. The binding reaction can be carried out in cold room for over two hours or at room temperature for 1 hour without significant difference.
  • TnT Quick coupled SP6 in vitro transcription and translation
  • IPR plates After binding, the IPR plates are washed twice with PBS and 100 ⁇ l of ORIGEN Assay Buffer (Cat. # 110006, IGEN International, Inc., Gaithersburg, MD) is added to each well before reading the ECL signal.
  • the ECL signals are measured using an imaging plate reader (IPR).
  • EXAMPLE 17 ECL Based Assays For DNA And Protein Interaction (Prophetic).
  • Glucocortcoid receptor p53 [SEQ ID No. 19] p53 YY1 [SEQ ID No. 20] YY1 Transcription factor C/EBP [SEQ ID No. 21] CCAAT enhancer binding protein gamma HNF [SEQ ID No. 22] HNF-4
  • the SH residues at the terminus are added to the sequence to allow for covalent coupling of the DNA to a carrier protein as described in Example 16.
  • the conjugation reaction to BSA is carried out with a pool of the oligos to give rise to a mixed conjugate that achieves the same effect of combining the various oligos into a pool.
  • Plasmid DNAs from these various clones are prepared using the Qiagen miniprep kit (Qiagen, Los Angeles, CA). These DNAs are used to produce the proteins as described in the Materials and Methods and the each protein is transferred to a separate well of the 4 spot DNA:BSA coated and BSA blocked ECL plates. To these wells 50 to 100 ng/well of Sulfo-TAG labeled Streptavidin (see example 2) is added as the detection agent. The binding reaction can be carried out in cold room for over two hours or at room temperature for 1 hour without significant difference. After binding, the ECL plates are washed twice with PBS and 100 ⁇ l of ORIGEN Assay Buffer (Cat. # 110006, IGEN International, Inc., Gaithersburg, MD) is added to each well before reading the ECL signal. The ECL signals are measured using an imaging plate reader (IPR).
  • IPR imaging plate reader
  • the peptide from IkBa, H2N-LKKERLLDDRHDS(p)GLDS(p)MKDEEYEC [SEQ ID No.1] is synthesized chemically.
  • the ECL label is coupled post synthesis using ORI-TAG-Maleimide (IGEN International, Inc., Gaithersburg MD) to produce the following peptide H2N-LKKERLLDDRHDS(p)GLDS(p)MKDEEYEC-ORI-TAG.
  • the following clones for testing are obtained from Incyte (CA) ⁇ TRCP1 IMAGE ID #3491843; ⁇ TRCP2 IMAGE ID #4237375; Grb2 IMAGE ID #4398016; PLC ⁇ IMAGE ID #4419252. These clones are grown up and DNA prepared using the Qiagen miniprep kit (Qiagen, Los Angeles, CA). These DNAs are used to produce the proteins in the transcription and translation reaction as described in the Materials and Methods.
  • the reaction products are added to separate wells of an IPR plate coated with streptavidin and combined with 10-100 ng/ml of ORI-TAG labeled peptide.
  • the plate is left on a tabletop shaker for 1 hour to allow for biotinylated proteins to bind to the surface of the plate and for the ORI-TAG labeled peptide to bind to biotinylated proteins. Thereafter the plate is washed three times with PBS followed by addition of 100 ⁇ l of ORIGEN Assay Buffer (IGEN International, Inc.; Gaithersburg, MD) into each well, and ECL signals are measured in IPR Analyzer. The results demonstrate specific signal, above that of the control clones, for the clones able to direct the transcription and translation of the bTRCP binding proteins (3491843 and 4237375).
  • EXAMPLE 19 Assay For The Detection Of Proteins Binding To Non-Peptide Small Molecule Binding Partners.
  • ECL labeled steroids are purchased from IGEN International, Inc. (Gaithersburg, MD, these are part of Roche Diagnostic Kits): Estradiol (2.75ng/ml), Progesterone (10ng/ml), Testosterone (3ng/ml), Cortisol (25ng/ml). These steroids are labeled via a peptide linker attached to the steroid.
  • the following clones for testing are obtained from Incyte (CA) Glucocortcoid receptor IMAGE ID # 4036433, 3584269; progesterone binding proteins IMAGE ID # 4449225, 4365385; and the control clones bTRCP1 IMAGE ID #3491843; bTRCP2 IMAGE ID #4237375;. These clones are grown up and DNA prepared using the Qiagen miniprep kit (Qiagen, Los Angeles, CA). These DNAs are used to produce the proteins as described in Example 7 and mixed with 1-10 ng/ml ORI-TAG-labeled steroid in IPR plate coated with streptavidin.
  • CA Incyte
  • the plate is left on a tabletop shaker for 1 hour to allow for biotinylated proteins to bind to the surface of the plate and for the ORI-TAG-labeled peptide to bind to biotinylated proteins. Thereafter the plate is washed three times with PBS followed by addition of 100 ⁇ l of ORIGEN Assay Buffer (IGEN International, Inc., Gaithersburg MD) into each well, and ECL signals are measured. The ECL signals are measured using an imaging plate reader (IPR). The results demonstrate specific signal above that of the control clones for the clones able to direct the transcription and translation of the steroid receptors. In addition, no specific signal is associated with related ECL labeled steroids that were not bound by the specific steroid receptors.
  • IPR imaging plate reader
  • the Sulfo-TAG labeled anti-BODIPY®FL antibody (Molecular Probes, CA) was produced and purified as described in Materials and Methods. The antibody is stored in 25mM TRIS-HCl pH 7.5, 0.05% sodium azide.
  • the following clones for testing are obtained from Incyte (CA) mouse fos IMAGE ID #,3583544, 4457761, 3586465; human fos IMAGE ID # 3688670; mouse jun IMAGE ID # 3493248, 3968444,4018781; human Jun IMAGE ID # 3606344,4053956,4446852, 3912000,4182406; and the control clones bTRCP1 IMAGE ID #3491843; bTRCP2 IMAGE ID #4237375;. These clones are grown up and DNA prepared using the Qiagen miniprep kit (Qiagen, Los Angeles, CA).
  • DNAs are used to produce the proteins encoded by the various cloned DNAs in the following transcription and translation reaction. These clones are transcribed and translated in two reactions one with biotin-lys-tRNA and one with FluoroTect TM Green(BODIPY®FL)-Lys-tRNA to generate a copy of each protein encoded by these clones labeled with biotin and a copy labeled with BODIPY®FL.
  • Transcription-translation reaction mixtures described in Materials and Methods were used to produce protein. Following these TnT reactions a sample from each clone from the biotin tRNA reaction is mixed with a sample from each clone from the FluoroTect TM Green-Lys-tRNA reaction as follows: Then 4-8 ⁇ l of the biotin-lys-tRNA TnT reaction and 4-8ul of the FluoroTect TM Green-Lys-tRNA TnT reaction is mixed with 50 ⁇ l of binding buffer (PBS, pH7.4, 0.1%BSA, 0.1% bovine IgG, 0.2% tween-20, and protease inhibitor tablets without EDTA (Roche)), and 50 ng Sulfo-TAG labeled anti-BODIPY®FL antibody in a IPR plate coated with streptavidin.
  • binding buffer PBS, pH7.4, 0.1%BSA, 0.1% bovine IgG, 0.2% tween-20, and protease inhibitor
  • This combination of the clones results in a set of incubations where each clone is allowed to bind to each of the other clones and also to its self from a complementary reaction with an alternatively modified tRNA.
  • the plate is left on a tabletop shaker for 1 hour to allow for biotinylated proteins to bind to the surface of the plate and for the Sulfo-TAG labeled antibody to bind to the BODIPY®FL labeled proteins. Thereafter the plate is washed three times with PBS followed by addition of 100 ⁇ l of ORIGEN Assay Buffer (IGEN International, Inc., Gaithersburg MD) into each well, and ECL signals are measured. The ECL signals are measured using an imaging plate reader (IPR). The results demonstrate specific signal above that of the control clones for the clones able to direct the transcription and translation of the proteins known to bind to each other.
  • IPR imaging plate reader
  • the following clones for testing are obtained pGBKT7-53, pGADT7-T, pGBKT7-c-Myc, pGADT7-Max and the control clones, pGBKT7, pGADT7 pGBKT7-lam (Clontech, Palo Alto CA). These clones were grown up and DNA prepared using the Qiagen miniprep kit (Qiagen, Los Angeles, CA). These DNAs are used to produce the proteins encoded by the various cloned DNAs in the following transcription and translation reaction.
  • Each of these clones are transcribed and translated, as described in Materials and Methods, in two reactions: one supplemented with biotin-lys-tRNA and one under normal conditions to generate a copy of each protein encoded by these clones labeled with biotin or unlabeled.
  • a sample from each clone from the biotin tRNA reaction is mixed with a sample from each clone from the standard TnT reaction as follows: 4-8 ⁇ l of the biotin-lys-tRNA TnT reaction and 4-8 ⁇ l of the standard TnT reaction is mixed with 50 ⁇ l of binding buffer (PBS, pH7.4, 0.1%BSA, 0.1% bovine IgG, 0.2% tween-20, and protease inhibitor tablets without EDTA (Roche)), and 50 to 100 ng/well of Sulfo-TAG labeled Streptavidin (see Materials and Methods) in NPT IPR plate coated with anti HA epitope antibody F-7 (Santa Cruz, CA).
  • binding buffer PBS, pH7.4, 0.1%BSA, 0.1% bovine IgG, 0.2% tween-20, and protease inhibitor tablets without EDTA (Roche)
  • Sulfo-TAG labeled Streptavidin see Materials and Methods
  • EXAMPLE 22 Assay To Detect And Identify Antigens Recognized By Antibodies.
  • clones are grown in 2 ml LB with 50 ⁇ g/ml Amp, and DNA is isolated using QIAprep Spin Miniprep Kit (Qiagen). DNA yield is 5-20 ⁇ g.
  • the clones are as follows; Sjogren syndrome antigen A2 (60kD, ribonucleoprotein autoantigen SS-A/Ro), 4395342; Sjogren syndrome antigen A1 (52kD, ribonucleoprotein autoantigen SS-A/Ro), 3922229, 3904700; Sjogren's syndrome nuclear autoantigen 1, 3459939, 3047963; Sjogren's syndrome/scleroderma autoantigen 1, 3857068,4342257; Sjogren syndrome antigen B (autoantigen La), 3454454, 3910607; ⁇ TRCP1, 3491843; ⁇ TRCP2, 4237375.
  • the DNA from these clones is translated in a transcription and translation system as described in Materials and Methods. Then 5 ⁇ l of the reaction product is mixed with 50 ⁇ l of binding buffer in a well of a 96 well NPT IPR plate coated with streptavidin . In this way 2, 96 well plates are generated with 5 rows A, B, C, D, E containing the 11 I clones in each of these rows, one clone in each well with the 12th well in each row as a control well (no protein) in combination with the two ⁇ TRCP clones, as control proteins. The plate is left on a tabletop shaker for 1 hour to allow for the biotinylated proteins from the transcription translation reaction to bind to the surface of the plate. Thereafter the plate is washed three times with PBS.
  • patient samples 1-10 are diluted into PBS, pH7.4, 0.3%BSA, 0.2% tween-20 to generate dilutions from 1/200 to 1/2,000 these diluted patient samples are then added 50 ⁇ l per well in such a way that each patient sample is added to a row on the 96 well plate with the 11 clones coated on to the surface. These plates are then left on a tabletop shaker for 1 hour to allow for the patient antibodies to bind to the proteins coated on the plate. Thereafter the plate is washed three times with PBS.
  • EXAMPLE 23 Demonstration Of A Multiwell Screening Method For Multiple Clones; Screen Of 90 EST Clones In 96-Well Format.
  • the example demonstrates a screen to identify (i) substrates of ubiquitylation in reticulocyte lysates; (ii) proteins binding to specific phosphopeptides; and (iii) proteins possessing tyrosine-kinase activities.
  • the clone IMAGE ID # were as follows; 3870426, 3893964, 3919253, 3491843, 4237375, 4398016, 4419252, 3907115, 2899921, 2900118, 2900273, 2900421, 2900551, 2900956, 2901296, 3048141, 3048356, 3048606, 3445488, 3446518, 3446546, 3449836, 3449955, 3450936, 3451168, 3452024, 3452494, 3452714, 3453890, 3454025, 3454207, 3454505, 3454625, 3455258, 3455871 3458308, 3458973, 3459189, 3459274, 3460054, 3460621, 3461825, 3462090, 3462906, 3463478, 3463640, 3464547, 4181547, 3845821, 3847788, 3853408, 3857590, 3857760, 38
  • clones were purified separately using Qiagen Midiprep kit and introduced into the 96-well plate at 470 ⁇ g/ml in addition to the rest of the clones to serve as positive controls in the assays.
  • Two of these control clones (IMAGE IDs 3920860, 3912628) contained full-length RGS4 cDNA, a known substrate of ubiquitylation (Examples 1, 2, 3, 4, 5, 6) and the remaining two clones (IMAGE ID 3491843 and 4237375) encode for ⁇ TRCP1 and ⁇ TRCP2 respectively, proteins known to bind to a phosphorylated peptide derived from IkBa (Examples 10, 11, 12).
  • Transcription-translation reactions were assembled in a 96-well plate as described in Materials and Method in a total volume of 19 ⁇ l.
  • the amounts of DNA in each reaction varied depending on DNA yields at purification stage, while the four positive control wells contained reactions with 20 ⁇ g/ml plasmid DNA.
  • the reactions were incubated at 30°C for 40min.
  • FIG. 2 shows that three RGS4-encoding IMAGE clones (3912628, 3914731, and 3920860) produced strong specific signal in this assay ( ⁇ ) comparable to the signal produced by the two control RGS4 clones ( ⁇ ). The rest of the 88 EST clones analyzed in this experiment produced much weaker signals ( ⁇ ). The assay was able to identify RGS4 as an efficient substrate of ubiquitylation in reticulocyte lysates, as previously reported ( Davydov and Varshavsky, 2000, J. Biol. Chem., 275:22931-22941 ).
  • a screen was conducted using 7-spot IPR plates.
  • the 7 spot NPT IPR plates are described in more detail in copending Provisional Application No. 60/301,932 (entitled “Assay Plates, Reader Systems and Methods for Luminescence Test Measurements", filed on June 29, 2001, hereby incorporated by reference) and particularly in the description of Plate Type D in Example 6.1 (NPT IPR plate) and U.S. Application Serial Nos. 10/185,274 and 10/185,363, filed June 28, 2002 , each hereby incorporated by reference.
  • These plates consist of a 96 wells with 7 zones (spots) for the immobilization of proteins or other reagents.
  • Each spot was coated with BSA conjugated to either a single phosphopeptide or multiple phosphopeptides.
  • the plates were prepared essentially as described in example 12. The seven spots supported, respectively, the following BSA conjugates IkB, pY992, pY1068, pY1148, Myc, "A”, and "6-P".
  • A refers to a conjugate of BSA with multiple peptides: IkB, pY1148, Myc which were then coupled to BSA.
  • 6-P refers to a conjugate of BSA with the following multiple peptides: IkB, pY1148, pY992, pY1068, Smad3C.
  • the assay 4 ul from TnT reaction product described above were added to the coated plate containing 50 ⁇ l of binding buffer supplemented with 50 ng/well Ru-Streptavidin. The binding reaction was allowed to proceed at room temperature for 1 hour with constant shaking. At the end of incubation, the plate was washed twice with PB5 and 100 ⁇ l of Origen Assay Buffer (IGEN International, Inc., Gaithersburg MD) was added for reading the ECL. The ECL signals were measured using an imaging plate reader (IPR). The raw data from the IPR reader was then normalized using two steps of normalization. The first round was based on the 50 lowest values for a given specific spot.
  • IPR imaging plate reader
  • results below demonstrate the expected correlation between the specific peptide and its binding partner demonstrating the value of this approach for the screening of libraries of clones for specific binding partners using a multi well plate with multiple binding domains (spots).
  • the results from the multiple peptide spots (“A” and "6-P") agreed with the single peptide spots demonstrating the value of pooling peptides to increase the number of peptides per screen.
  • the Myc peptide showed binding to the F-Box protein ⁇ TRCP indicating that it might be modulated via ubiquitination as IkBa is on its phosphorylation.
  • results from the screen were as expected.
  • results for the top 9 clones for each of the single peptide spots are shown below.
  • the data from the multiple peptide spots also agreed with this data. This approach with multiple spots readily allowed us to normalize the background within a well which prove valuable with some clones that had high background binding to all the peptide spots in the well.
  • Immobilized protein was then used to phosphorylate substrate protein, poly Glu-Tyr (Sigma, pEY) in a 50 ⁇ l reaction containing 5 nM pEY, 25 mM Tris buffer pH 7.4, 5 mM MgCl 2 , 0.05 mM Na 3 VO 4 , 0.004% TritonX-100, 2 mM DTT, EDTA-free protease inhibitor (Roche Molecular Biochemicals) and 100 mM ATP, by shaking at room temperature for 60 minutes.
  • poly Glu-Tyr Sigma, pEY
  • a 50 ⁇ l reaction containing 5 nM pEY, 25 mM Tris buffer pH 7.4, 5 mM MgCl 2 , 0.05 mM Na 3 VO 4 , 0.004% TritonX-100, 2 mM DTT, EDTA-free protease inhibitor (Roche Molecular Biochemicals) and 100 mM ATP, by shaking at room temperature for
  • the unbound reagents were removed by washing three times with water, and the TAG label ECL was detected in presence of 100 ⁇ l IPR assay buffer (IGEN International, Inc., Gaithersburg MD).
  • the ECL signals were measured using an imaging plate reader (IPR).
  • FIG. 3 shows that two Tyrosine Kinase encoding clones (IMAGE ID # 3870426 and 3893964) produced strong specific signals in this assay ( ⁇ ) when compared to the remaining EST clones which produced much weaker signals ( ⁇ ).
  • the assay was able to identify specific tyrosine kinase activity toward substrate pEY from the immobilized proteins.
  • EXAMPLE 24 Direct Coating Of Proteins Onto Magnetic Beads.
  • EST clones were obtained from Incyte (Palo Alto, CA). Plasmid DNA was prepared from these EST clones using QIAprep96 Turbo Miniprep (cat#27191, QIAgen, Los Angeles, CA), according to manufacturer's instructions. cDNA was eluted using 150 ⁇ l buffer EB (QIAgen). The DNA from these clones was translated in a transcription and translation system as described in Materials and Methods. For binding of the proteins from the TnT reaction to the magnetic beads we used the following protocol.
  • the transcription-translation reaction (1 ⁇ l) was added to 40 ⁇ g of uncoated magnetic beads (SPHERO(TM) Carboxyl Magnetic Particles- Smooth Surface, 1% w/v, 3.2 ⁇ m Cat# CMS-30-5, Spherotech,) in 50ul PBS pH 7.4, with EDTA and protease inhibitors (Roche Biochemicals) in a 96 well plate.
  • the beads were incubated for 2 hours followed by the addition of 35-40ng Sulfo-TAG labeled streptavidin to each well in a 50 ⁇ l volume using assay buffer (25mM Tris pH7.4, 0.005% Triton, 1% BSA, protease inhibitors) and the plate was incubated for 1h at room temperature with shaking.
  • EXAMPLE 25 Screening for EGFR Binding Proteins.
  • the example describes an ORIGEN based HTS assay for EGFR binding to reticulocyte lysate-expressed Grb2 and PLC ⁇ .
  • the assay uses Sulfo-TAG labeled anti-EGFR/anti-phosphotyrosine for detection.
  • the bound EGFR was directly detected in the original plate by incubating with 20 nM TAG labeled anti-EGFR (Clone 225, Neo Markers, CA) in 50 ⁇ l buffer containing 25 mM Tris-HCl buffer pH 7.4, 5 mM MgCl 2 , 0.05 mM Na 3 VO 4 , 0.004% TritonX-100, 2 mM DTT and 0.05% IgG at room temperature for 60 min.
  • the unbound reagents were removed by one time washing with TT- buffer [25 mM Tris-HCl buffer pH 7.4 and 0.004% Triton], and the TAG label was detected in presence of 150 ⁇ l IPR assay buffer (0.4 mM Gly-Gly buffer, pH 7.8, 1 mM EDTA, 0.1 M TPA).
  • TT- buffer 25 mM Tris-HCl buffer pH 7.4 and 0.004% Triton
  • Grb2 and PLC ⁇ specifically bind to EGFR; these are suggested to interact through phosphorylated sites of EGFR.
  • the control protein Luciferase (Mock) did not show any significant level of binding to EGFR.
  • the binding of EGFR to Grb2 or PLC ⁇ was detected either by identifying tyrosine kinase activity of EGFR towards pEY (see Table 32) or directly by using TAG-labeled anti-EGFR (Table-33).
  • the detection of bound EGFR was substantially increased by its prolonged (12 hr) phosphorylation of tyrosine kinase substrate pEY (see Table 32).
  • EXAMPLE 26 ORIGEN Based HTS Assay For Protein Tyrosine Phosphatase Activity Of PTP-N2 (Protein Tyrosine Phosphatase, Non-Receptor Type 2) Expressed In Reticulocyte Lysate.
  • the example demonstrates an assay to screen for protein tyrosine phosphatase activity.
  • PTPs Protein Tyrosine phosphatases
  • T-cell protein tyrosine phosphatase is one such phosphatase that exits the nucleus upon EGF receptor activation and recognizes the EGFR and p52S Shc as a cellular substrates ( Tiganis et. Al Mol. Cell. Biol. 18, 1680, 1998 ).
  • the phosphorylated poly(Glu,Tyr) was used as a substrate to measure the phosphatase activity of the purified and immobilized PTP-N2.
  • a reaction mixture containing 5 nM phospho-pEY (described above), 5 mM MgCl 2 , 25 mM Tris-HCl buffer pH 7.4, 0.004% TritonX-100, 2 mM DTT and EDTA-free protease inhibitor (Roche Molecular Biochemicals). This mixture was incubated with shaking at room temperature 45 min to allow dephosphorylation of the phospho-pEY substrate by the immobilized phosphatase. Following this incubation 45 ⁇ l of this mixture was transferred to a streptavidin-coated NPT IPR plate and the solution analyzed for phospho-poly(Glu,Tyr) content as described above.
  • This example illustrates the use of methods of the invention to express a library of potential kinases and screen this library for kinase activity.
  • the kinases were assayed for their ability to auto-phosphorylate as well as their ability to phosphorylate a non-autologous substrate.
  • Proteins were produced in an in vitro transcription and translation reaction as described in the Materials and Methods. This process resulted in the transcription and translation of the various proteins with biotin groups randomly incorporated at some lysine residues.
  • the reaction was stopped by mixing with BB2 buffer.
  • Biotinylated proteins from 50 ⁇ l of the mixture (representing 0.5 ⁇ l to 5 ⁇ l of TnT reaction) were immobilized onto streptavidin or avidin coated NPT-IPR plates by shaking at room temperature for 45 min. The activity of the immobilized protein towards pEY or autophosphorylation was further assayed by the methods described in examples 7 and 8.
  • EXAMPLE 28 ORIGEN Based HTS Assay For Kinase Activity Of Tyrosine Kinase Array In Presence Of Kinase Inhibitors.
  • This example shows the profiling of a panel of kinase inhibitors against a panel of tyrosine kinases.
  • the example demonstrates the use of proteins expressed in in vitro transcription/translation systems in inhibition assays and/or drug screening assays.
  • the example shows specificity data for proteins generated from a library of eighteen full-length clones.
  • the assay format developed for this purpose utilizes following steps: Proteins were produced using 100 ng of plasmid DNA Mock (Luciferase, Promega) or tyrosine kinases (Fert2 IMAGE ID # 4485050, FGFR1 IMAGE ID # 4037899, Fgfr2 IMAGE ID # 2654352, Src IMAGE ID # 3156400, BMX IMAGE ID # 3919253, LCK IMAGE ID # 4191610, Epha7 IMAGE ID # 3991628, Ephb4 IMAGE ID # 4013934, Tek IMAGE ID # 3482498, HCK IMAGE ID # 4778819, BTK IMAGE ID # 4478463, CSK IMAGE ID # 4398416, SYK IMAGE ID # 3870426, FGR IMAGE ID # 4419973, PTK2B IMAGE ID # 4339456, FRK IMAGE ID # 4515877, Kit IMAGE ID #
  • TT- buffer [25 mM Tris-HCl buffer pH 7.4 and 0.004% Triton]
  • high-salt wash buffer 25 mM Tris-HCl buffer pH 7.4, 0.004% Triton, 1M NaCl and 5 mM DTT] and 3 washes with TT-buffer.
  • the mixture was incubated at room temperature for 10 min before starting the kinase reaction by adding 25 ⁇ l of 200 ⁇ M ATP in buffer A. This mixture was incubated with intermittent shaking at room temperature for 2 hours to overnight to allow (a) phosphorylation of the pEY substrate and (b) autophosphorylation of the immobilized kinases. Autophosphorylation and phosphorylation of the pEY was measured as described in Examples 7 and 8.

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